Endometriosis-related markers and uses thereof

ABSTRACT

The present invention relates to markers of endometriosis which are differentially expressed in the endometrial cells of females with endometriosis compared to endometriosis-free females. The invention also relates to methods for determining likelihood of endometriosis in female subjects, to methods for grading endometriosis in females suffering from endometriosis and to methods for treating this disease. The invention is also concerned with polynucleotides, probes, primers and kits useful for reducing into practice the above-mentioned methods which are more rapid, non invasive, much less complicated and much less costly than laparoscopy.

This application is a divisional patent application of U.S. patentapplication Ser. No. 09/794,928, filed Feb. 26, 2001 and now issued asU.S. Pat. No. 6,777,182 B2, which claims the benefit of U.S. provisionalapplications No. 60/185,063 filed on Feb. 25, 2000 and No. 60/225,745filed on Aug. 17, 2000.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to markers of endometriosis and moreparticularly to methods for determining likelihood of endometriosis infemale subjects, to methods for grading endometriosis in femalessuffering from endometriosis and to methods for treating this disease.The invention is also concerned with polynucleotides, probes, primersand kits useful for reducing into practice the above-mentioned methods.

b) Brief Description of the Prior Art

Endometriosis is one of the most common gynecological disorders,affecting up to 10-15% of women of reproductive age. It is mainlyassociated with severe pelvic pain and/or infertility, but also withdysmenorrhea, dyspareunia, and several other symptoms such asintraperitoneal bleeding, back pain, constipation and/or diarrhea.Endometriosis is characterized by the implantation and growth ofendometrial cells (which normally constitute the lining of the uterus)in extra-uterine sites, most frequently in the peritoneal cavity. Theseverity of the disease can be graded. According the American Society ofReproductive Medicine (ASRM), the disease is classified in four stages,namely, minimal (stage I), mild (stage II), moderate (stage II), andsevere (stage IV). Although the etiology and pathogenesis ofendometriosis remain unclear, the theory of retrograde menstruation isthe most widely accepted to explain the presence of endometrial cells inectopic sites. However, retrograde menstruation occurs in most women.Thus, a certain genetic potential or predisposition, present in theendometrial cells, might be responsible for the presence of the disease.Initially, this genetic potential may relate to mutations in the genome,but in addition, it may also lead to subsequent altered gene expression.

At present, direct visualization of the endometriotic lesions undersurgical procedures (laparoscopy or laparotomy) is the only reliablemethod to diagnose endometriosis. However, this method is highlyinvasive (i.e. surgery under general anesthesia) and costly. The periodof time between the onset of symptoms and disease diagnosis can be aslong as 8 to 12 years. Ideally, the prospect to diagnose endometriosismore easily, rapidly, and as early as possible during the course of thedisease would definitely reduce the number of years during whichpatients endure pain, infertility or other symptoms.

Based on this perspective, several investigators have sought to identifybiological markers (proteinic and genetic) that could efficiently beused as predictive tools for endometriosis. However, to date, no one hasbeen able to do so.

For instance, several proteins have also been shown to be differentiallyexpressed in endometriosis. These include the tissue inhibitor ofmetaloproteinase-1 (TIMP-1), α_(v)β₃ integrin, MCP-1, aromatase P450 andplasminogen activator-receptor and inhibitors. Unfortunately, theclinical relevance of these markers is uncertain since diagnosticparameters such as sensitivity and specificity of these candidatemarkers are still poorly defined.

Bcl-2 has been reported to be upregulated during the proliferative phaseof the ovarian cycle in the eutopic endometrium of diseased women(Meresman et al. (2000) Fertil. Steril. 74(4): 760-6) as well as inendometriotic lesions in both phases of the cycle (Jones et al. (1998)Hum. Reprod. 13(12): 3496-502) and in macrophages from the peritonealfluid of women having endometriosis (McLaren et al. (1997) Hum. Reprod.12(1): 146-52). However, these results differ from our data presentedherein in which a downregulation of Bcl-2 is observed in the eutopictissue of women with endometriosis compared to disease-free women,independent of the phase of the ovarian cycle.

Connexin 43 (Cx43), a protein involved in gap junctions, has beenreported to be aberrantly expressed in the glandular uterine epitheliumof ectopic endometrial tissue in women with endometriosis (Regidor et al(1997) Mol. Hum. Reprod. 3:375-381). The goal of this study was solelyto determine the hormonal regulation of connexins in endometriotictissues, and consequently, this report did not analyze eutopic tissue ineither women with endometriosis nor in disease-free women. Thus,findings in this study have little clinical or diagnostic relevance.

Human cyclooxygenase-2 (COX-2) is involved in prostaglandin synthesis,and, as a result, has been implicated in the growth and differentiationof endometrial stromal cells as well as during the implantation periodnecessary to establish pregnancy (Marions and Danielsson (1999) Mol.Hum. Reprod. 5:961-5). Due to its role in implantation during pregnancy,it has been postulated that COX-2 may be involved in the implantation ofendometrial cells in ectopic sites, giving rise to endometriosis.However, to date, there have not been any conclusive reportsdemonstrating the role of COX-2 in endometriosis, nor that an alterationof its expression leads to the disease.

An increase in the expression at the protein level of heat shock protein70 (HSP70) has been described in endometrial glandular cells of womenhaving endometriosis and adenomyosis compared to a control group (Ota etal. (1997) Fertil Steril 68: 23-28). This result was obtained byimmunohistochemistry. The same authors using the same technique showedthat endothelial nitric oxide synthase (eNOS) and superoxide dismutase(SOD) were also up-regulated in the endometrium of patients withendometriosis or adenomyosis (Ota et al. (1998) Fertil Steril 69:303-308; Ota et al. (1999) Fertil Steril 72: 129-134). However, thesestudies have limited clinical value because some of the experiments werenot always carried out with an accurate technical approach, and becausethe markers were tested on a small number of patients yielding nostatistically significant results. Furthermore, in Ota's studies,altered gene expression was found to occur in the ectopic tissue, asopposed to in the eutopic endometrium, and the results presented aretherefore not industrially applicable.

Others groups studying gene expression have reported that thegluthatione S-transferase (GST) gene had a higher degree of polymorphismin endometriosis compared to a control group, and therefore representedan overall less-performing detoxification system which predisposed womento the disease (Baranova et al., (1999) Mol. Hum. Reprod. 5:636-641).These results reflect a genetic predisposition to have the diseaserather than the likelihood of endometriosis.

Overall, no one has ever described any methods for determining thelikelihood of endometriosis in females, any methods for efficientlyidentifying females suffering from endometriosis, nor any methods forgrading endometriosis in females suffering from the disease.

There is therefore a need for an alternative approach to laparoscopy orlaparotomy to diagnose and determine the stage of endometriosis. Moreparticularly, it would be highly desirable to be provided with methodswherein endometrial cells samples are assayed for expression levels ofspecific endometriosis-related genes (RNA or cDNA transcripts or theircorresponding proteins), that are known to be differentially expressedin the endometrial cells of females with endometriosis (Endo group)compared to endometriosis-free females (Control group).

The present invention fulfils these needs and also other needs whichwill be apparent to those skilled in the art upon reading the followingspecification.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a method for determiningthe likelihood of endometriosis in female subjects.

The present invention also aims to provide a method for gradingendometriosis in females suffering from this disease, as well as amethod for treating this disease.

The invention is also concerned with primers, probes and kits useful forreducing into practice the above-mentioned methods.

In accordance with an aspect of the present invention, there is provideda method for determining the likelihood of endometriosis in femalesubjects whereby expression levels of one or more selectedendometriosis-related marker(s) is assayed, and the expression level ofthe marker is indicative of the likelihood of endometriosis in thesubject. In a preferred embodiment, the endometriosis-related marker(s)is selected from the group consisting of:

-   -   i) genes selected from the group consisting of the genes listed        in TABLE 1 for which a GENBANK™ gene name is given;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NOs: 1            to 16; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii).

In another aspect, the present invention relates to the used ofOXPHOS-endometriosis-related markers in a method for determininglikelihood of endometriosis in a female subject. This method comprisesthe steps of:

-   -   obtaining a sample of eutopic endometrial cells, preferably        epithelial endometrial cells, from the female subject;    -   assaying the endometrial cells sample for the expression level        of at least one endometriosis-related marker involved in the        oxidative phosphorylation pathway and/or the internal redox        potential sensors pathway (OXPHOS) of the endometrial cells;    -   determining likelihood of endometriosis in the female subject by        comparing the expression level for the at least one        OXPHOS-endometriosis-related marker to an established baseline        level for the at least OXPHOS-endometriosis-related marker.

In a further aspect, the present invention relates to the assay ofnucleic acid product(s) of endometriosis-related marker(s) in methodsfor determining likelihood of endometriosis in a female subject. Thismethod comprises the steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from the female subject;    -   assaying the endometrial cells sample for the expression level        of at least one nucleic acid product of an endometriosis-related        marker selected from the group consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and    -   determining likelihood of endometriosis in the female subject by        comparing the expression level of said at least one nucleic acid        product to an established baseline level.

In another aspect, the present invention relates to the assay of proteinproduct(s) of endometriosis-related marker(s) in methods for determininglikelihood of endometriosis in a female subject. This method comprisesthe steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from the female subject;    -   assaying this endometrial cells sample for the expression level        of at least one protein product of an endometriosis-related        marker or of least one fragment of this protein product, the at        least one endometriosis-related marker being selected from the        group consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and    -   determining likelihood of endometriosis in the female subject by        comparing the expression level of the at least one protein        product to an established baseline level.

According to another aspect, the present invention relates to a methodfor grading endometriosis. This method comprises the steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from a female subject suffering from        endometriosis; and    -   assaying said endometrial cells sample for the expression level        of at least one endometriosis-related marker selected from the        group consisting of:        -   i) genes selected from the group consisting of RNA helicase,            NADH dehydrogenase, hUCC1, AK3, GST, 12S rRNA, CO2,            aconitase, c-jun, Cx43, GPx4, cox2, hUCC1, Glut-1, TI227H,            IL-1β, HSP 90;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO                10 and SEQ ID NO 13; and            -   b) fragments of the ribonucleic acids defined in a); and        -   iii) peptides or proteins encoded by the genes defined in            i), or encoded by the ribonucleic acids defined in ii);            the expression level of the at least one            endometriosis-related marker being indicative of the stage            of endometriosis in the female subject.

According to another aspect, the present invention relates to a kit fordetermining likelihood of endometriosis in a female subject or forgrading endometriosis in a female subject suffering from endometriosis.The kit comprises at least one binding molecule for binding to nucleicacid product(s) or protein product(s) of endometriosis-relatedmarker(s). According to a preferred embodiment, the binding moleculebinding molecule is a nucleic acid. In another preferred embodiment, thebinding molecule is an isolated antibody.

According to a further aspect, the present invention relates isolatedpolynucleotides, primers and/or probes and to their uses in methods fordetermining likelihood of endometriosis in a female subject or forgrading endometriosis in a female subject suffering from endometriosis.

The present invention also relates to a method for treatingendometriosis comprising the step of modulating expression level of atleast one selected endometriosis-related marker.

An advantage of the present invention is that it is rapid, non-invasive,and significantly less complicated and costly than performinglaparoscopy or laparotomy. In contrast to currently-available methods,it is possible, according to the present invention, to directly measureexpression levels of endometriosis-related genes that are expresseddifferentially in endometrial cells depending of the presence/absence ofendometriosis and the stage of the disease with relatively high levelsof sensitivity and specificity.

Other objects and advantages of the present invention will be apparentupon reading the following non-restrictive description.

DETAILED DESCRIPTION OF THE INVENTION A) Definitions

Throughout the text, the words “base pairs” are generally abbreviated as“bp”, the words “deoxyribonucleic acid” as “DNA”, the words “ribonucleicacid” as “RNA”, the words “complementary DNA” as “cDNA”, the words“polymerase chain reaction” as “PCR”, and the words “reversetranscription” as “RT”. Nucleotide sequences are written in the 5′ to 3′orientation unless stated otherwise.

In order to provide an even clearer and more consistent understanding ofthe specification and the claims, including the scope given herein tosuch terms, the following definitions are provided:

Binding molecule: Any molecule which adheres to a given target moleculein a adequate manner, for example, enzymes to substrates, antibodies toantigens, or a DNA strand to its complementary strand.

Derived from: As used herein, a cDNA “derived from” a gene when thiscDNA has been synthesized from a messenger RNA coded by this gene.Typically, cDNAs are synthesized in an in vitro reaction catalyzed by aviral reverse transcriptase in which a complementary strand of DNA isproduced from an isolated mRNA template.

Endometrial cells: Refer to the cells which form the tissue lining theuterus (stromal and epithelial cells). Normally, endometrial cells aresloughed off during the woman's menstrual period, and afterwards growsback and slowly thickens until the next period. As used herein,“endometrial cells” encompasses eutopic as well as ectopic endometrialcells, where endometrial cells that usually constitute the lining of theuterine cavity are considered eutopic, and those outside the uterus areconsidered ectopic.

Endometriosis-related marker: Refers to any amino acid product ornucleic acid product for which the level of expression in theendometrium of a female is correlated with endometriosis.

Expression level: Refers to the amount of a definite amino acid productor of a definite nucleic acid product in a given cell or tissue.“Expression” more particularly refers to the process by which a genecoded information is converted into the structures present and operatingin the cell. As used herein, expressed genes include those that aretranscribed into mRNA and then translated into protein and those thatare transcribed into RNA but not translated into protein (for example,transfer and ribosomal RNAs). Similarly, the expression “baselineexpression level” refers to the level of expression which is found undernormal conditions and normal level of functioning (e.g.endometriosis-free female). The terms “overexpression” and“underexpression” refer to an upward or a downward deviationrespectively in assayed levels of expression as compared to the baselineexpression level.

Female subject: Refers to human females being in reproductive age.According to the present invention, the female subject would preferablypresents clinical symptoms of endometriosis such as infertility andpelvic pain.

Fragment: Refers to a section of a molecule, such as a protein or anucleic acid, and is meant to refer to any portion of the amino acid ornucleotide sequence.

Gene: Refers to a DNA sequence related to a single polypeptide chain orprotein, and as used herein includes the 5′ and 3′ untranslated regions.

Giving rise to: As used herein, a ribonucleic acid “gives rise to” acDNA when it serves as a template for synthesizing the cDNA. Typically,cDNAs are synthesized in an in vitro reaction catalyzed by a viralreverse transcriptase in which a complementary strand of DNA is producedfrom an isolated mRNA template.

Likelihood: As used herein in combination with the term “endometriosis”,it more particularly refers to an existing probability of a femalesubject of really suffering from endometriosis. It does not refer to apredisposition to suffering in the future from the disease.

Nucleic acid product: Any molecule having one or more nucleotide(s)derived from the expression of gene. As used herein in combination withthe expression “endometriosis-related marker”, it more particularlyrefers to RNA and fragments thereof, cDNAs and fragments thereof, andexpressed sequence tags (ESTs) which expression is correlated with theexpression of an endometriosis-related marker (see hereinbefore).

OXPHOS: Abbreviation for “oxidative phosphorylation”. As used herein, itrefers to the sequence of enzymatic reactions coupled to themitochondrial respiratory chain whereby ATP is phosphorylated, as wellas to the nucleic acid and protein products involved in these reactions.

Phase of estrous cycle: refers to the period of estrus in whichphysiological changes occur in females as a result of hormonalinfluences during the menstrual or ovarian cycle. Briefly, In humanfemales, the menstrual cycle is divided into two phases, namely, the“proliferative phase” (also called the follicular phase, herein referredto as P phase) and the “secretory phase” (also called the luteal phase,herein referred to as S phase). The proliferative phase normally extendsfrom day 0 to day 14 of the menstrual cycle, and the secretory phasenormally extends from day 15 to day 28, where ovulation occurs on day 14of a standard menstrual cycle.

Protein product: refers to organic molecules that contain two or moreamino acids which are assembled by formation of peptide bonds duringribosomal translation of a messenger RNA. As used herein in combinationwith the expression “endometriosis-related marker”, it more particularlyrefers to peptides, proteins, glycoproteins, and protein fragments whoseexpression is correlated with the expression of an endometriosis-relatedmarker (see hereinbefore).

B) General Overview of the Invention

The present invention concerns the early detection, diagnosis,prognosis, grading and treatment of endometriosis. Markers ofendometriosis in the form of nucleic acid sequences, proteins andpeptides isolated from human endometrial cells are disclosed. Levels ofexpression of these “endometriosis-related markers” are indicative ofthe likelihood of endometriosis in a female subject and of the stage ofendometriosis in a female subject diagnosed as having endometriosis.Preferably, the endometriosis-related markers of the invention are thoselisted in Table 1 hereafter. Table 2 lists the nucleotide sequences ofsome of these endometriosis-related markers, also referred by theinventors as DD1 to DD16 (SEQ ID NOs: 1 to 16).

C) Methods for Determining the Likelihood of Endometriosis

i) Endometriosis-Related Markers

According to an aspect of the invention, the endometriosis-relatedmarkers are used in a method for determining the likelihood ofendometriosis in a female subject. This method comprises the steps of:

-   -   obtaining a sample of endometrial cells from a female subject        (preferably eutopic endometrial cells, and even more preferably        epithelial eutopic endometrial cells);    -   assaying the endometrial cells sample for the expression level        of at least one endometriosis-related markers selected from the        group consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and        -   iii) peptides or proteins encoded by the genes defined in            i), or encoded by the ribonucleic acids defined in ii).

According to this method, the expression level of theendometriosis-related marker(s) is indicative of the likelihood ofendometriosis in the female subject. Preferably, the method furthercomprises the step of comparing the expression level for theendometriosis-related marker to an established baseline expressionlevel. The baseline level for the endometriosis-related marker(s) ispreferably established by assaying the expression level for the samemarker(s) in a negative reference group of endometriosis-free women.

According to a preferred embodiment, overexpression of at least oneendometriosis-related marker listed in Table 4 is indicative of a higherlikelihood of endometriosis in the female subject, and moreparticularly, the following endometriosis-related markers:

-   -   i) genes selected from the group consisting of: NADH        dehydrogenase, hUCC1, Paralemmin, citrate transport protein,        HIF1α, ARNT, Glut-1, MnSOD, GPx, ATP synthase, c-jun, Cx43, HSP        70, and cox2;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NO: 9,            SEQ ID NO: 10, SEQ ID NOs: 13 to 15; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii);

According to another preferred embodiment, underexpression of at leastone endometriosis-related marker listed in Table 5 is indicative of ahigher likelihood of endometriosis in the female subject, and moreparticularly, the following endometriosis-related markers:

-   -   i) genes selected from the group consisting of: Cap43, RNA        helicase, CO3, FKHR, 12S rRNA, AK3, catalase, GST, eNOS, 12S        rRNA, TI227H, CO2, aconitase, ANT-1, Bcl-2, COUP-TF, IL-1β, HSP        90, GPx4, and GRP78;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NOs: 1            to 8, SEQ ID NO 11, SEQ ID NO 12, and SEQ ID NO 16; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii).

The inventors have also found that some endometriosis-related markersare modulated depending on whether endometrial cells are obtained at theproliferative phase or at the secretory phase of the estrous cycle ofthe female subject. Tables 6 and 7 hereafter provide a list of theendometriosis-related markers of the invention whose expression levelswere found to be modulated in the proliferative and the secretoryphases, respectively. Therefore, in a preferred embodiment, the methodof the invention further comprises the steps of: i) defining at whichphase of the estrous cycle the endometrial cells were obtained; and ii)selecting the endometriosis-related marker for which expression level isto be assayed according to the phase defined in i). The phase of theestrous cycle may be evaluated by using techniques well known in the artsuch as histological examination (preferably of endometrial tissues),methods for evaluating level of expression of RNA, and methods forevaluating sex steroids levels to name a few.

Therefore, in a preferred embodiment, the endometrial cells from thefemale subject are sampled at the proliferative phase of her estrouscycle, and overexpression level of at least one endometriosis-relatedmarker selected from the group consisting of:

-   -   i) genes selected from the group consisting of: NADH        dehydrogenase, hUCC1, Paralemmin, citrate transport protein,        HIF1α, ARNT, Glut-1, MnSOD, GPx, ATP synthase, c-jun, Cx43, HSP        70, and cox2;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NO: 9,            SEQ ID NO: 10, SEQ ID NOs: 13 to 15; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii);        is indicative of a higher likelihood of endometriosis in said        female subject as compared to an endometriosis-free female        subject.

In another preferred embodiment, the endometrial cells from the femalesubject are sampled at the secretory phase of her estrous cycle, andoverexpression level of at least one endometriosis-related markerselected from the group consisting of:

-   -   i) genes selected from the group consisting of hUCC1,        Paralemmin, citrate transport protein, HIF1α, Glut-1, MnSOD,        GPx, ATP synthase, c-jun, Cx43, HSP 70, and cox2;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NOs: 13            to 15; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii);        is indicative of a higher likelihood of endometriosis in said        female subject as compared to an endometriosis-free female        subject.

Yet, in a further preferred embodiment, the endometrial cells from thefemale subject are sampled at the proliferative phase of her estrouscycle, and underexpression level of at least one endometriosis-relatedmarker selected from the group consisting of:

-   -   i) genes selected from the group consisting of: Cap43, RNA        helicase, FKHR, 12S rRNA, AK3, GST, eNOS, TI227H, CO2, ANT-1,        Bcl-2, IL-1β, and HSP 90;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NO: 2,            SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID            NO 12, and SEQ ID NO 16; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii);        is indicative of a higher likelihood of endometriosis in said        female subject as compared to an endometriosis-free female        subject.

In another preferred embodiment, the endometrial cells from the femalesubject are sampled at the secretory phase of her estrous cycle, andunderexpression level of at least one endometriosis-related markerselected from the group consisting of:

-   -   i) genes selected from the group consisting of: RNA helicase,        CO3, FKHR, 12S rRNA, AK3, catalase, GST, 12S rRNA, TI227H,        aconitase, Bcl-2, COUP-TF, IL-1β, HSP 90, GPx4, and GRP78;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NOs: 1            to 8, SEQ ID NO 11, SEQ ID NO 12, and SEQ ID NO 16; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii);        is indicative of a higher likelihood of endometriosis in said        female subject as compared to an endometriosis-free female        subject.

In another preferred embodiment of the invention, the expression levelof at least two endometriosis-related markers is assayed in combination.By combining markers, it is generally possible to increase thespecificity and the sensitivity of the methods of the invention. Table11 contains some examples of endometriosis-related markers combination.Methods for assaying the expression levels of genetic and proteinicmarkers such as the endometriosis-related markers of the presentinvention are well known. Methods and materials for assaying nucleicacids include: BioChips, membranes, and glass cDNA array-basedtechniques; RT-PCR, in situ hybridization; in vitro promoter-fusionstudies in cell lines or primary culture models; transcription ratestudy techniques such as nuclear run-on; membrane blot hybridizationapproaches; direct labeling of the nucleic acids. Methods and materialsfor assaying proteins and peptides include: membrane blot hybridizationapproaches; direct labeling of the protein or peptide; proteomics; flowcytometry; immunocytochemistry; immunohistochemistry; and ELISA-basedapproaches. A person skilled in molecular biology and immunology willknow how to select, adapt, and use these methods according to hisspecific need in order to obtain valuable results. For example, it couldbe relatively easy to develop biochips bearing genetic markersconsidered the best in terms of specificity and sensitivity for cDNAhybridization arrays to screen females, and more particularly femaleshaving endometriosis-related symptoms.

In some cases, overexpression of certain genetic and proteinic markersinduces the production of auto-antibodies in the patients' blood.Therefore, measurement of these auto-antibodies could be anotheralternative, although indirect, to assay the expression levels of thegenetic and proteinic markers according to the invention.

ii) OXPHOS-Endometriosis-Related Markers

In another aspect, the present invention relates to the use ofOXPHOS-endometriosis-related markers in a method for determining thelikelihood of endometriosis in a female subject. OXPHOS refers to theoxidative phosphorylation pathway and/or the internal redox potentialsensors pathway of the endometrial cells. This method comprises thesteps of:

-   -   obtaining a sample of eutopic endometrial cells, preferably        epitelial endometrial cells, from the female subject;    -   assaying the endometrial cell sample for the expression level of        at least one endometriosis-related marker involved in OXPHOS;    -   determining the likelihood of endometriosis in the female        subject by comparing the expression level for the at least one        OXPHOS-endometriosis-related marker to an established baseline        level for the at least OXPHOS-endometriosis-related marker.

According to a preferred embodiment, the OXPHOS-endometriosis-relatedmarker involved in the oxidative phosphorylation pathway of theendometrial cells is a gene selected from the group consisting of NADHdehydrogenase, citrate transport protein, HIF1α, ARNT, AK3, Glut-1,MnSOD, GPx, GRP78, catalase, GST, eNOS, CO2, aconitase, ANT-1, ATPsynthase, Bcl-2, GPx4, and cox2. It may also be preferable to select theOXPHOS-endometriosis-related marker to be assayed according to the phaseof the estrous cycle in which the endometrial cells belong.

iii) Nucleic Acid Products of an Endometriosis-Related Marker

In another aspect, the present invention relates to assaying nucleicacid products of an endometriosis-related marker according to methodsfor determining the likelihood of endometriosis in a female subject.This method comprises the steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from the female subject;    -   assaying the endometrial cells sample for the expression level        of at least one nucleic acid product of an endometriosis-related        marker selected from the group consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and    -   determining likelihood of endometriosis in the female subject by        comparing the expression level of said at least one nucleic acid        product to an established baseline level.

According to a preferred embodiment, the likelihood of endometriosis inthe female subject is determined by assaying the level of expression ofat least one nucleic acid product of a gene selected from the groupconsisting of: Cap43, RNA helicase, NADH dehydrogenase, CO3, FKHR,hUCC1, Paralemmin, citrate transport protein, HIF1α, ARNT, AK3, MnSOD,GPx, aconitase, ATP synthase, c-jun, COUP-TF, Cx43, HSP 70, and GRP78.

According to another preferred embodiment, the likelihood ofendometriosis in the female subject is determined by assaying the levelof expression of a ribonucleic acid selected from the group consistingof: ribonucleic acids giving rise to cDNAs comprising a sequenceselected from SEQ ID NOs: 8 to 16 and fragments thereof.

More preferably, the baseline level of the selectedendometriosis-related marker(s) is established by assaying itsexpression level in a negative reference group of endometriosis-freewomen. Preferably also, the at least one nucleic acid product is amessenger ribonucleic acid (mRNA) and this mRNA serves as a template forthe synthesis of a cDNA.

The expression level of the at least one nucleic acid product can beassayed using well known methods such as biochips, membranes, and glasscDNA array-based methods; RT-PCR; in situ hybridization; in vitropromoter-fusion studies in cell lines or primary cultures; transcriptionrate study methods; membrane blot hybridization; and labeling. Even morepreferably, the expression level for at least two endometriosis-relatedmarkers is assayed. It may also be desirable to define at which phase ofthe estrous cycle the endometrial cells were obtained in order toproperly select the endometriosis-related marker for which a nucleicacid product to be assayed is in accordance with this phase.

iv) Protein Products of an Endometriosis-Related Marker

In another aspect, the present invention relates to assaying amino acidproducts of an endometriosis-related marker according to methods fordetermining the likelihood of endometriosis in a female subject. Thismethod comprises the steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from the female subject;    -   assaying this endometrial cells sample for the expression level        of at least one protein product of an endometriosis-related        marker or of at least one fragment of this protein product, the        at least one endometriosis-related marker being selected from        the group consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and    -   determining the likelihood of endometriosis in the female        subject by comparing the expression level of the at least one        protein product to an established baseline level.

According to a preferred embodiment, the baseline level of the selectedprotein product(s) is established by assaying its expression level in anegative reference group of endometriosis-free women. Preferably also,the at least one protein product is the CAP-43 protein or a fragmentthereof.

The expression level of the at least one protein product can be assayedusing well known methods such as membrane blot hybridization; labeling;proteomics; flow cytometry; immunocytochemistry; immunohistochemistryand ELISA. Even more preferably, the expression level of at least twoendometriosis-related markers is assayed in combination. It may also bepreferable to define at which phase of the estrous cycle the endometrialcells were obtained in order to properly select theendometriosis-related marker so that the protein product to be assayedis in accordance with this phase.

D) Methods for Grading Endometriosis

The present inventors also found that the expression levels of specificendometriosis-related markers were modulated depending on the stage ofthe disease. Table 8 hereafter provides a list of theendometriosis-related markers whose expression levels were found to bemodulated depending on the stage of the disease. Therefore, according toanother aspect, the present invention relates to a method for gradingendometriosis. This method comprises the steps of:

-   -   obtaining a sample of endometrial cells, preferably eutopic        endometrial cells, from a female subject suffering from        endometriosis; and    -   assaying said endometrial cells sample for the expression level        of at least one endometriosis-related marker selected from the        group consisting of:        -   i) genes selected from the group consisting of RNA helicase,            NADH dehydrogenase, hUCC1, AK3, GST, 12S rRNA, CO2,            aconitase, c-jun, Cx43, GPx4, cox2, hUCC1, Glut-1, TI227H,            IL-1β, HSP 90;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO                10 and SEQ ID NO 13; and            -   b) fragments of the ribonucleic acids defined in a); and        -   iii) peptides or proteins encoded by the genes defined in            i), or encoded by the ribonucleic acids defined in ii);            the expression level of the at least one            endometriosis-related marker being indicative of the stage            of endometriosis in the female subject.

In a preferred embodiment, the method is used so that the level ofexpression of the at least one endometriosis-related marker indicateswhether the female subject is at stage I or II, or at stage III or IV ofendometriosis.

More particularly, it was found that overexpression of NADHdehydrogenase, c-jun, Cx43, and/or cox2 is indicative of whether thefemale subject is substantially at stage I or II of endometriosis; andthat overexpression of hUCC1 and/or Glut-1 is indicative of whether thefemale subject is at stage III or IV of endometriosis. It was also foundthat underexpression of CO2, SEQ ID NO:1, SEQ ID NO:6, aconitase, GPx4,RNA helicase, AK3, GST, and/or 12S rRNA is indicative of whether thefemale subject is substantially at stage I or II of endometriosis; andthat underexpression of TI227H, IL-1β, and/or HSP 90 is indicative ofwhether the female subject is at stage III or IV of endometriosis.Whether these markers are underexpressed or overexpressed is thus astrong indication of the stage of the disease.

More preferably, the method further comprises the step of comparing theexpression level for the at least one endometriosis-related marker to anestablished baseline level. The baseline level may be obtained by:

-   -   assaying the expression level of the at least one        endometriosis-related marker in a sample of endometrial cells        from a first positive reference group of female subjects known        to be at a substantially definite stage of endometriosis;    -   assaying the expression level of the at least one        endometriosis-related marker in a sample of endometrial cells        from a second positive reference group of female subjects known        to be at another substantially definite stage of endometriosis        different from the stage of the first positive reference group;        and    -   comparing expression level of the at least one        endometriosis-related marker with expression levels from the        first and second positive reference groups.

Measurement of the expression levels of the endometriosis-relatedmarkers (genetic or protein) of the present invention can beaccomplished as described herein. It may also be preferable to define atwhich phase of the estrous cycle the endometrial cells were obtained inorder to properly select the endometriosis-related marker so that theprotein product to be assayed is in accordance with this phase.

E) Kit

According to another aspect, the present invention relates to a kit fordetermining likelihood of endometriosis in a female subject or forgrading endometriosis in a female subject suffering from the disease.The kit of the invention comprises:

-   -   at least one binding molecule for binding to a nucleic acid        product or a protein product of an endometriosis-related marker,        the endometriosis-related marker being selected from the group        consisting of:        -   i) genes selected from the group consisting of the genes            listed in TABLE 1 for which a GENBANK™ gene name is given;        -   ii) ribonucleic acids selected from the group consisting of:            -   a) ribonucleic acids giving rise to cDNAs selected from                the group consisting of: cDNAs derived from genes                defined in i); and cDNAs comprising a sequence selected                from SEQ ID NOs: 1 to 16; and            -   b) fragments of the ribonucleic acids defined in a); and        -   i) peptides or proteins encoded by the genes defined in i),            or encoded by the ribonucleic acids defined in ii); and    -   at least one element selected from the group consisting of: a        support for the binding molecule(s), mixing tubes, buffers,        enzymes, and substances for the detection of the binding        molecule(s).

According to a preferred embodiment, the binding molecule is a nucleicacid hybridizing under standard conditions to a nucleic acid selectedfrom the group consisting of:

-   -   a) ESTs and mRNAss for which a GENBANK™ accession number is        given in TABLE 1;    -   b) single-stranded nucleic acids which hybridize under standard        conditions to the nucleic acids defined in a);    -   c) single-stranded nucleic acids obtained by reverse        transcription of a nucleic acid defined in a) or b);    -   d) DNAs and cDNAs for which a GENBANK™ accession number is given        in TABLE 1;    -   e) double-stranded and single-stranded nucleic acids which        hybridize under standard conditions to the nucleic acids defined        in d);    -   f) double-stranded and single-stranded nucleic acids obtained by        reverse transcription of a nucleic acid defined in d) or e); and    -   g) fragments of the nucleic acids defined in a) to f).

As used herein, nucleic acid hybridization refers to the principle thattwo single-stranded nucleic acid molecules that have complementary basesequences will reform the thermodynamically favored double-strandedstructure if they are mixed under proper conditions. For example, amedium stringency condition could be provided by approximately 0.1 to0.25M NaCl at temperatures of approximately 37° C. to 55° C., while alow stringency condition could be provided by approximately 0.15 M to0.9 M NaCl, at temperatures ranging from approximately 20° C. to 55° C.Thus, the “standard hybridization conditions” varies depending on thedesired results.

According to another preferred embodiment, the binding molecule is anisolated antibody directed against at least one protein product of anendometriosis-related marker as defined previously, or directed againsta fragment of the protein product(s).

Other types of binding molecules include small molecules such as sugarsand glycoproteins.

F) Polynucleotides, Primers, Probes and their Uses

According to another aspect, the present invention relates to isolatedpolynucleotides. The polynucleotides of the invention are selected fromthe group consisting of:

-   -   a) polynucleotides comprising a nucleic acid sequence selected        from the group consisting of: SEQ ID NOs: 1 to 10, SEQ ID NOs:        12 to 16, and portion thereof; and    -   b) polynucleotides comprising a nucleic acid sequence        complementary to the polynucleotides defined in a).

These polynucleotides have many uses, particularly as biological markersaccording to the methods of the present invention, as primers and/or asprobes for endometriosis. They could also be used as a template forpreparing single-stranded or double-stranded nucleic acids useful ingene therapy methods (antisense, gene silencing, double-stranded RNA,etc).

The invention also relates to primers and/or probes for determininglikelihood of endometriosis in a female subject or for gradingendometriosis in a female subject suffering from endometriosis.According to the invention, the primer or the probe comprises anisolated nucleic acid selected from the group consisting of:

-   -   a) ESTs and mRNAs for which a GENBANK™ accession number is given        in TABLE 1;    -   b) single-stranded nucleic acids which hybridize under standard        conditions to the nucleic acids defined in a);    -   c) single-stranded nucleic acids obtained by reverse        transcription of a nucleic acid defined in a) or b);    -   d) DNAs and cDNAs for which a GENBANK™ accession number is given        in TABLE 1;    -   e) double-stranded and single-stranded nucleic acids which        hybridize under standard conditions to the nucleic acids defined        in d);    -   f) double-stranded and single-stranded nucleic acids obtained by        reverse transcription of a nucleic acid defined in d) or e); and    -   g) fragments of the nucleic acids defined in a) to f).

The invention also encompasses the uses of the aforementioned isolatedpolynucleotides and nucleic acids in methods for determining likelihoodof endometriosis in a female subject or in methods for gradingendometriosis in a female subject suffering from endometriosis. Thesemethods may include the use of biochips, membranes, and glass cDNAarray-based techniques; RT-PCR; in situ hybridization; in vitropromoter-fusion studies in cell lines or primary cultures; transcriptionrate study methods; membrane blot hybridization; and labeling. Forinstance, some of the isolated nucleic acids described above considered“the best” in terms of specificity and sensitivity to screen femalescould be coupled to a biochip for screening females for endometriosisand/or for evaluating the stage of her disease.

The invention also includes diagnostic method for the detection ofendometriosis in a female subject. The diagnostic method of theinvention comprises the use of any of the aforementioned methods for thedetermination of the likelihood, and/or the aforementioned kits,isolated polynucleotides, nucleic acids probes and primers. Depending onthe selected endometriosis-related markers, particularly if used incombination, it is indeed possible according to the present invention todiagnose females having endometriosis with a very high sensibility and avery high specificity.

G) Method for Treating Endometriosis

According to a further aspect, the present invention relates to a methodfor treating endometriosis. This method comprises the step of modulatingthe expression level of an endometriosis-related marker selected fromthe group consisting of:

-   -   i) genes selected from the group consisting of the genes listed        in TABLE 1 for which a GENBANK™ gene name is given;    -   ii) ribonucleic acids selected from the group consisting of:        -   a) ribonucleic acids giving rise to cDNAs selected from the            group consisting of: cDNAs derived from genes defined in i);            and cDNAs comprising a sequence selected from SEQ ID NOs: 1            to 16; and        -   b) fragments of the ribonucleic acids defined in a); and    -   iii) peptides or proteins encoded by the genes defined in i), or        encoded by the ribonucleic acids defined in ii).

In a preferred embodiment, the expression level of at least one of theendometriosis-related markers listed in Table 5 is increased. In anotherpreferred embodiment, the expression level of at least one of thefollowing endometriosis-related markers listed in Table 4 is decreased.

Methods and materials for increasing or decreasing the expression levelsof genetic and proteinic markers such as the endometriosis-relatedmarkers of the present invention are well known and within the skill ofa person in the art. A non-limitative list of known methods andmaterials includes: diet, vitamins, dietary supplements, gene therapymethods, antisense oligonucleotides, drugs and hormonal medications.

For instance, it is hypothesized that compounds which decrease ROSlevels in cells such as ROS scavengers molecules commercialized byMETAPHORE PHARMACEUTICALS™ (www.metaphore.com) not actually used fortreating endometriosis could be used according to the methods oftreatment of the present invention. Therefore, pharmaceuticalcompositions for treating endometriosis and comprising substance(s)modulating the expression level of the aforementionedendometriosis-related marker(s) are also within the scope of the presentinvention.

EXAMPLES

The following examples illustrate the wide range of potentialapplications of the present invention and are not intended to limit itsscope. Modifications and variations can be made therein withoutdeparting from the spirit and scope of the invention. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice for testing the present invention, thepreferred methods and materials are described.

Example 1 Genetic Markers

1) Problematic

In the absence of any reliable and non-invasive diagnostic tool for thedetection of endometriosis, the focus was placed on the identificationof genes differentially expressed in the endometrium of female subjectssuffering from endometriosis (Endo group) when compared toendometriosis-free subjects (Control group). To achieve this, threequantitative approaches were used, namely differential display, cDNAarray hybridization, and specific RT-PCR. Unless specified otherwise,all experiments have been performed using RNA obtained from the enrichedglandular fraction of the endometrium. In addition, for some analyses,results were also obtained from RNA isolated from unfractionedendometrial biopsies.

2) Materials and Methods

The following are experimental procedures and materials that were usedfor the example set forth below.

Patient Recruitment

Common prerequisites for patients to be enrolled in the study carried onfor the present example, both in the endometriosis-free control andendometriosis-positive groups, were the following:

-   -   premenopausal age;    -   not currently menstruating;    -   menstrual cycles between 21 and 35 days;    -   no acute salpingitis;    -   no HIV, hepatitis B or C positive diagnosis;    -   not pregnant or having been pregnant in the last three months;    -   not currently breastfeeding;    -   no use of a compound selected from the group consisting of GnRH        agonists, Progestins, Danazol, and oral contraceptives in the        last three months; and    -   no use of intra-uterine device (I.U.D.) in the last three        months.

Women recruited in the endometriosis-positive group who providedbiopsies of the endometrium were selected among women undergoinglaparoscopy and for whom the presence of endometriosis was confirmed atthe time of surgical examination. Stages of the disease were definedaccording to the American Society of Reproductive Medicine (ASRM)classification as follows: stage I (minimal), stage II (mild), stage III(moderate), and stage IV (severe).

To be eligible in the control group, women who provided biopsies had tofulfill the following conditions:

-   -   undergo laparoscopy for tubal ligation or tubal reanastomosis;    -   no endometriosis lesion detected in the peritoneal cavity at        surgery;    -   no history of endometriosis in first-degree relatives; and    -   no infertility indication.

Women with endometriosis (Endo) were subdivided in two experimentalgroups, namely, EXP 1 (stages I and II) and EXP 2 (stages III and IV).Experimental groups were further subdivided into proliferative (P) phaseand secretory (S) phase groups based on the phase of the menstrual cycleindicated by last menstruations and confirmed by histologicalexamination.

Tissue Samples

Biopsies of the endometrium were obtained from patients under anesthesiaprior to laparoscopy with a conventional curette. Harvested tissue wasmaintained on ice and used for the experiment within four hours.Experiments were performed either on enriched glandular fractions or onunfractioned biopsies.

Isolation and Preparation of RNA from Enriched Glandular Fraction

Glandular fractions from the endometrium were enriched by enzymaticdigestion. The tissue was first cut into small pieces in the presence ofHBSS medium (LIFE TECHNOLOGIES™). The pieces were then digested with anenzymatic mixture containing 3.4 mg/ml pancreatin, 0.1 mg/mlhyaluronidase and 1.6 mg/ml collagenase (SIGMA™) for 50 min at 37° C.under 5% CO₂. Following enzymatic digestion, glandular cells wereseparated from the stromal fraction by size exclusion. That is, thedigested cell suspension was subjected to successive rounds offiltration through 41 μm and 11 μm filters. The glandular fractionconsists of cells that were retained on the filters.

Total RNA was isolated from endometrial glandular cells. The glandularcell fraction was weighed, and cells were resuspended at a finalconcentration of 100 mg/ml of a denaturing solution containing 2.7 Mguanidine thiocyanate, 1.3 M ammonium thiocyanate, and 0.1 M sodiumacetate, pH 4.0. The suspension was then extracted twice withphenol/chloroform before precipitation with 1 volume of isopropanol. TheRNA pellet was washed with 80% ethanol, and resuspended in H₂O at afinal concentration of 1 μg/μl.

Isolation and Preparation of RNA from Unfractioned Biopsies

Total cellular RNA was isolated directly from endometrial biopsy tissue.Briefly, 200-300 mg of biopsy tissue was homogenized using an electrichomogenizer in the presence of 3 ml of TRIZOL™ (LIFE TECHNOLOGIES™). RNAwas prepared according to the TRIZOL™ manufacturer's protocol. Usually,2 or 3 additional extractions with phenol/chloroform were necessary toobtain a clear interface. RNA was then precipitated and resuspended asdescribed above.

Differential Display

Differential display (DD) was performed using total RNA isolated fromglandular cells or unfractioned biopsies according to Liang and Pardee(Differential Display: Methods and Protocols, 1997, Humana Press,Totowa, N.J. p. 1-11). Amplifications were done in a STRATAGENEROBOCYCLER™ (STRATAGENE™) involving 40 cycles of 94° C. (1 min), 40° C.(2 min), 72° C. (1 min) with a final extension of 7 minutes at 72° C.The following reagents were used: M-MLV Reverse Transcriptase (LIFETECHNOLOGIES™), ³³P-dATP (AMERSHAM PHARMACIA BIOTECH™, 2500 Ci/mmol),and rTaq DNA Polymerase (AMERSHAM PHARMACIA BIOTECH™).

Cloning of PCR Products

Fragments identified by differential display were cloned using the TACLONING KIT™ (INVITROGEN™) according to the manufacturer's instructions.Clones containing the desired fragments were identified by colony-PCR asdescribed in Maniatis et al. (Maniatis et al., 1992, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) and using the lacZ −20 and reverse primers.

Reverse-Northern Blot

Colony-PCR products were deposited on positively-charged nylon membranes(BOEHRINGER MANNHEIM™) and hybridized with ³²P-labeled differentialdisplay probes. Several clones with the expected differential patternwere selected for sequencing. Clones exhibiting identical sequences wereaccepted as candidates.

Sequencing

Sequencing was performed using the ABI PRISM RHODAMINE CYCLE SEQUENCINGKIT™ (PE APPLIED BIOSYSTEMS™) and run on an automated ABI PRISM 310™Sequencer (PE APPLIED BIOSYSTEMS™).

cDNA Microarrays

Expression of genes was studied using duplicate membranes of the HUMANand CANCER ATLAS cDNA ARRAYS™ (CLONTECH LABORATORIES™). Glandular RNA (3μg) from control individuals and patients at the same phase of themenstrual cycle were reverse transcribed in presence of α³²P-dATP(AMERSHAM PHARMACIA BIOTECH™, 3000 Ci/mmol). Hybridizations and analyseswere performed according to the user's manual provided by themanufacturer.

RT-PCR

Total cellular RNA, previously isolated from glandular cells orunfractioned biopsies as described above, was used as a template for theproduction of cDNA. Briefly, 1 μg of RNA was digested with 1 U DNAse 1(LIFE TECHNOLOGIES™) according to the manufacturer's instructions.Digested RNA was then reverse transcribed into cDNA using 200 U M-MLVReverse Transcriptase in the presence of 0.4 μM random primers (LIFETECHNOLOGIES™), 2 mM of each of dGTP, dATP, dTTP, and dCTP, and reactionbuffer supplied. Following incubation at 37° C. for 1 hour, the reactionwas terminated by boiling for 5 minutes. PCR amplification of cDNA wasperformed according to standard protocols described in Maniatis et al.(Maniatis et al., supra). cDNAs were first normalized by a series of PCRamplifications of the internal control gene, glyceraldehyde phosphatedehydrogenase (GAPDH).

PCR reactions contained 5 all of cDNA (standardized by GAPDH), 0.5 μM ofeach of two specific oligonucleotide primers, 0.2 mM of each of dGTP,dATP, dTTP, and dCTP, 1 U rTaq DNA Polymerase, and reaction buffersupplied. Amplifications were performed in a BIOMETRA UNO II™ or TGRADIENT THERMOCYCLER™ (BIOMETRA™) involving 25-40 cycles of 94° C. (45seconds), a specific annealing temperature (45 seconds), 72° C. (1 min.)with a final extension of 7 minutes at 72° C. Annealing temperaturesvaried according to the specific oligonucleotide primers used, while thenumber of cycles varied according to the abundance of the cDNA templatein question.

Southern Blot

Southern blots were performed according to standard protocols describedin Maniatis et al. (Maniatis et al., Supra). Briefly, following specificPCR amplification of cDNAs described above, samples were separated on1.5% agarose gels, and transferred onto BIODYNE™ 0.45 μm membranes (PALLCORPORATION™) according to the manufacturer's instructions. Themembranes were fixed by UV irradiation, and hybridized overnight at 65°C. with specific plasmid probes which were ³²P-labeled using a randomprimer labeling kit (MEGAPRIME™ DNA labeling system, AMERSHAM PHARMACIABIOTECH™). Products were visualized by autoradiography, scanned, andanalyzed by MOLECULAR ANALYST™ software (BIO-RAD™). Band intensitieswere equalized based on the intensity of an internal control (GAPDH).

Sequence Analysis

Nucleotide sequence alignments were performed with GENBANK™ through theBLAST software available at the National Center for BiotechnologyInformation (NCBI) website (www.ncbi.nih.gov). Given a weak percentageof sequence misreadings in our data and in GENBANK™, homologies of 90%or more were considered as an identity.

Statistical Analysis

Data was analyzed using MICROSOFT EXCEL™. For statistical analysis, theStudent-t Test was performed and results were considered significantwith a p value of less than or equal to 0.05.

Specificity is defined herein as the probability of absence of a diseasemarker in the Control group whereas sensitivity is the probability ofpresence of the disease marker in the Endometriosis group. Calculationsfor specificity and sensitivity are performed according to thefollowing. First, an arbitrary cut-off value is established on thegraphical representation of the sample (RNA, cDNA, protein, or other) inquestion. For samples that are upregulated in the diseased group,specificity is calculated by counting the number of patients in theControl group that are below the cut-off value, divided by the totalnumber of patients in that group and expressed as a percentage;sensitivity is calculated by counting the number of patients in theEndometriosis group that are above the same cut-off value, divided bythe total number of patients in that group and expressed as apercentage. For samples that are downregulated in the disease group,specificity is calculated by counted the number of patients in theControl group that are above the cut-off value, divided by the totalnumber of patients in that group and expressed as a percentage;sensitivity is calculated by counting the number of patients in theEndometriosis group that are below the same cut-off value, divided bythe total number of patients in that group and expressed as apercentage.

3) Results

I—Differential Display (DD)

Enriched glandular cells were isolated from endometrial biopsies andtotal RNA was extracted. RNA extracts in both the Control and Endogroups were divided into two subgroups according to the phase of themenstrual cycle, namely, proliferative (P) or secretory (S) phase.Initially, differential signals were selected based on their appearancein more than 50% of samples of one group compared to the other. Selectedsignals were purified from the gel and cloned in E. coli. Severalrecombinant plasmids for each signal were submitted to the validationstep by reverse-Northern blotting as described in the presentapplication. Nucleotide sequence was determined and analyzed fordistinct plasmids of each series, and specific primers were synthesizedfor the next step.

Expression level of the candidates was further analyzed bysemi-quantitative RT-PCR on different groups of RNA. Experiments basedon cycle number and cDNA amounts were performed to ensure linearity ofthe PCR reactions. Expression level of GAPDH was used as the internalcontrol to normalize measurements. The Endo group was subdivided in twoexperimental groups: EXP 1 included minimal and mild stages of thedisease (I and II) and EXP 2 consisted of the moderate and severe stages(III and IV) of endometriosis. Table 3 summarizes average values bygroup for all endometriosis-related markers analyzed.

Cut-off values (not shown) were selected to estimate the clinical valueof each marker, namely, specificity and sensitivity. Hence, for eachmarker, the cut-off value was determined to allow maximal specificityand sensitivity values. Specificity corresponds to the probability ofthe absence of a marker in the control group and sensitivity representsthe probability of the presence of the same marker in the patient group.

A list of all the cDNAs identified by the differential display approachis given herein. For each candidate, the GENBANK™ accession number ofhomologous genes is given in Table 1. Nucleotide sequence data andcorresponding SEQ ID NO are listed in Table 2.

The DD1 marker (SEQ ID NO: 1) shows 94% homology in GENBANK™ with a cDNAcloned from fetal kidney tissue. This cDNA sequence also displays 100%homology to a region in the 22q11 chromosome (AC007064). Expressionanalysis showed in a significant decrease in the secretory phase of theEXP 1 group compared with the secretory phase of the Control group(p=0.01) (Table 3). With a cut-off established at 0.4, a specificity of75% and a sensitivity of 90% were obtained (Table 5).

The DD2 marker (SEQ ID NO: 2) corresponds to an expression sequence tag(EST) encoding a phosphoprotein which has also been isolated bydifferential display from human glioma cells. The homology encompassesan upstream region of 40 bp (97%) and a downstream region of 89 bp(96%). A gap of 40 bp is present in the middle of the alignment betweenthe two sequences. The same alignment was obtained with the 1-mychomolog cDNA (HSLMYCH). This may be due to alternate splicing of acommon gene in different tissues, or two distinct genes of a givenfamily. Presence of this cDNA has never been described in theendometrium. As shown in Table 3, this gene transcription wasdownregulated in the Endo group (p=0.04). The cut-off value of 0.13gives 65% specificity and 77% sensitivity (Table 5).

The DD3 marker (SEQ ID NO: 3) showed a 100% homology with themitochondrial origin of replication (ori). To date, no mRNA-encodinggene has been mapped in this region of mitochondrial DNA. According tothe results obtained for the present example shown in Table 3,expression is markedly decreased in the Endo group (p=0.01). The markerparameters with a 0.7 cut-off value are 63% specificity and 69%sensitivity (Table 5).

The DD4 marker (SEQ ID NO: 4) showed 98% homology with a cDNA isolatedfrom human colon tumors. Up to date, no known function has beenassociated with this gene. As shown in Table 3, expression issignificantly decreased in the secretory phase of the Endo group(p=0.04). With a threshold of 1.3, the candidate gene has a specificityof 83% and a sensitivity of 85% (Table 5).

The DD5 marker (SEQ ID NO: 5) corresponds to a gene having various namessuch as Cap43, Drg1, TDD-5, rit 42, Ndr 1, Proxy-1 or RTP, the mostcommon and the one used herein being Cap43, Cap43 mRNA has a 1759 bp3′-untranslated region and its predicted open reading frame encodes a394 amino acid residue polypeptide. Previous studies have shown amodulated expression of the Cap43 mRNA in various types of cells, butnot in endometrial cells nor in endometriosis. As shown in Table 3, ageneral decrease in the proliferative phase of all the endometriosispatients (p=0.03) was observed. A cut-off value of 1.2 results in 78%specificity and 79% sensitivity (Table 5).

The sequence of the DD6 marker (SEQ ID NO: 6) matches at 97% of homologywith a genomic sequence file. In addition, the cDNA segment of DD6contains an alu sequence at the 3′ end. As shown in Table 3, asignificant decrease was observed between the Control and EXP 1 in thesecretory phase (p=0.018). A cut-off value of 0.4 yields the followingparameters: specificity 89%, sensitivity 100% for the EXP 1 (Table 5).

Several ESTs were identical to the sequence DD7 (SEQ ID NO: 7). Allknown sequences cloned from transformed tissues such as pancreasadenocarcinoma, prostatic intraepithelial neoplasia, or a moderatelydifferentiated endometrial carcinoma. The DD7 cDNA contains also an alusequence. As shown in Table 3, a severe decrease in gene expression wasobserved for this marker in the proliferative phase of the Endo group(p=0.019). Specificity of 80% and a sensitivity of 70% with a 0.5cut-off value were found (Table 5).

Sequence homologies indicated that the sequence of the DD8 marker (SEQID NO: 8) was identical to a gene cloned from Jurkat cells, brain, andother tissues. In Table 3, a slight but significant decrease in DD8expression in the EXP 1 group (p=0.02), with specificity and sensitivityvalues of 67% and 60% respectively, at a cut off value of 0.6, wasobserved (Table 5).

The DD9 marker (SEQ ID NO: 9) corresponds to the subunit III of themitochondrial enzyme, NADH dehydrogenase, which is involved in cellularoxygen metabolism. This gene plays a key role in the electron transportchain and energy production. As seen in Table 3, a substantial increasein the proliferative phase of the Endo group (EXP 1 and EXP 2) isdetected (p=0.008). A 0.2 cut-off value provides a specificity of 70%and a sensitivity of 59% (Table 4).

The DD10 marker (SEQ ID NO: 10) corresponds to the NADH dehydrogenasesubunit II. As with DD9, this gene product is part of the respiratoryenzyme complex localized in the mitochondria. The entire enzyme iscomposed of 12 different polypeptides. In Table 3, validation of thismarker showed a higher level of expression in the proliferative phase ofthe EXP I group (p=0.04). Diagnostic value of the gene was evaluatedwith a 0.2 cut-off value giving a specificity of 71% and a sensitivityof 71% (Table 4).

The DD11 marker (SEQ ID NO: 11) corresponds to the cytochrome oxidase(CO3) (described below).

The DD12 marker (SEQ ID NO: 12) corresponds to the transcription factorForkhead domain protein (FKHR). Its presence in endometrium has neverbeen reported in literature. In Table 3, expression analysis of thismarker on unfractioned endometrial tissue RNA showed a decrease ofexpression in the Endo group (p=0.015). The marker was evaluated with acut-off value of 23 with a specificity of 81% and sensitivity of 56%when both phases were considered (Table 5). When DD12 expression wasanalyzed by Northern blotting on unfractioned biopsy RNA samples, asignificant decrease in the Endo group was detected compared to thecontrols (p=0.05) (Table 5). Using a cut-off value of 0.4, thespecificity was 55% and the sensitivity was 68%. This result is inagreement with the inventor's initial observation using RT-PCR.

The DD13 marker (SEQ ID NO: 13) corresponds to hUCC1 gene messenger RNA.The same cDNA was previously cloned from colon cancer. In Table 3,expression analysis of this marker with unfractioned tissue RNA showed ahigher level of expression in the EXP 2 group (p=0.04). Diagnostic valueof the gene was evaluated with a 0.5 cut-off value giving a specificityof 62% and a sensitivity of 64% in the EXP 2 group (Table 4).

The sequence of the DD14 marker (SEQ ID NO: 14) is 98% identical to arecently cloned human cDNA encoding a putative membrane-boundmorphoregulatory protein which shows homology with paralemmin, amembrane-bound morphoregulatory protein. As seen in Table 3, an increasein the Endo group (EXP 1 and EXP 2) is detected with unfractioned biopsyRNA (p=0.04). A 1.2 cut-off value provides a specificity of 78% and asensitivity of 58%, when only the S-phase is considered (Table 4).

The DD15 marker (SEQ ID NO: 15) corresponds to the citrate transportprotein, one of the DiGeorge syndrome markers in the 22q11 region. Asshown in Table 3, this marker exhibits in unfractioned biopsy RNA asubstantial increase of expression in the Endo group (p=6.10⁻⁴). Aspecificity of 85% and a sensitivity of 79% were obtained with a cut-offvalue of 0.18 (Table 4).

The sequence of the DD16 marker (SEQ ID NO: 16) shows 90% homology withsome human ESTs and 82% homology with the human mitochondrial 12S rRNAgene. As shown in Table 3, the expression of this marker wasdown-regulated in the Endo group of unfractioned biopsy RNA (p=3×10⁻⁵).With a cut-off value of 2, this marker showed a specificity of 70% and asensitivity of 78% (Table 5).

II—Other Approaches

In the present example, another semi-quantitative technique, namelyreverse-Northern blotting with cDNA arrays, was used to identify othergenes that are differentially expressed in endometriosis. The finalvalidation analysis was done by standardized RT-PCR, similar to thedifferential display approach. Table 1 gives the known GENBANK™accession number for each gene which were, according to the presentinvention, identified as an endometriosis-related marker.

Hypoxia-induced factor 1α (HIF-1α) is one of the two subunits of atranscription factor involved in oxidative stress. As seen in Table 3,statistical analysis showed a significant increase of HIF-1α mRNA in theEndo group (p=0.028). With a cut-off value of 1.1, a specificity of 65%and a sensitivity of 72% were obtained. When HIF-1α expression wasanalyzed by Northern blotting on unfractioned biopsy RNA samples, asignificant increase in the P-phase of the Endo group was detectedcompared to the control P-phase (p=0.019) (Table 4). Using a cut-offvalue of 0.95, the specificity was 83% and the sensitivity was 80%. Thisresult is in agreement with the inventors' initial observation onepithelial cells RNA.

The aryl hydrocarbon receptor nuclear translocator (ARNT) (or HIF-1β) isthe second subunit of the hypoxia-induced transcription complex. Infact, ARNT has a dual role: it may be coupled to HIF-1α as in the caseof hypoxia, but it may also form a heterodimer with the aromatic (aryl)hydrocarbon receptor (AhR) to counteract toxic situations. Inendometriosis, the ARNT gene is transcribed at a higher rate than thatobserved for its partner, HIF-1α. As seen also in Table 3, a significantincrease in the proliferative phase patients of the Endo group (p=0.02)was registered. Specificity of this marker was 67% and sensitivity 83%with a cut-off value of 0.6 (Table 4). These parameters are comparablewith the ones for HIF-1α in the proliferative phase. Comparableexpression patterns for HIF-1α and ARNT is a strong argument in favor ofthe oxidative stress pathway (OXPHOS) involvement in endometriosis.

Adenylate kinase is a ubiquitous enzyme that contributes to thehomeostasis of the cellular adenine and guanine composition. Theadenylate kinase isozyme 3 (AK3) isoform is exclusively located in theinner membrane of mitochondria and its activity is involved in energytransfer. Its modulation has been associated with hypoxia and, morespecifically, with HIF1α activity. In Table 3, a severe down-regulationof AK3 expression was observed in the EXP 1 group (p=0.005). The cut-offof 0.75 produced a specificity of 68% and a sensitivity of 82% (Table5).

The glucose transporter isoform 1 (Glut-1) plays an important role incellular energy metabolism. As mentioned above, expression of Glut-1 isregulated by the HIF-1α/ARNT transcription factor complex. In addition,Glut-1 expression is induced in various circumstances such as oxidativestress or cellular transformation. In Table 3, a significantup-regulation in Glut-1 expression in the EXP 2 group compared to theControl group (p=0.037) was observed. Moreover, the specificity andsensitivity of Glut-1 are 71% and 67% respectively, when using a cut-offvalue of 1 (Table 4).

Cellular respiration occurring in the mitochondria results in theproduction of reactive oxygen species (ROS). Chronic ROS exposure canresult in permanent oxidative damage to cellular and mitochondrialproteins, lipids, and nucleic acids. To protect against oxidative damagecaused by these toxic products, mammalian cells have evolved protectivemechanisms. Detoxifying enzymes include manganese superoxide dismutase(MnSOD), glutathione peroxidase (GPx), catalase (CAT), and glutathioneS-transferase (GST). MnSOD is transcriptionnally up-regulated inmitochondria to detoxify superoxide anion (O₂ ⁻) by converting it tohydrogen peroxide (H₂O₂).

Analysis of MnSOD expression in Table 3 demonstrates a general andsignificant up-regulation in the entire Endo group compared to Controlgroup (p=0.017). With a cut-off of 0.9, the specificity of MnSOD was 76%and its sensitivity was 58%. Similar results were obtained withunfractioned biopsy RNA, and a significant elevation in the whole Endogroup was detected (p=0.003). With a cut-off value of 0.9, the markerexhibited a specificity of 81% and a sensitivity of 53% (Table 4).

As discussed above, GPx is involved in the ROS detoxification pathway inthe mitochondria. More specifically, GPx converts the H₂O₂ produced byMnSOD into H₂O. Statistical analysis showed a significant increase inGPx mRNA expression in the Endo group compared to Controls(p=1.7×10⁻¹¹), as shown in Table 3. This resulted in a specificity of100% and sensitivity of 87.5%, when using a cut-off value of 1.2 (Table4).

In extra-mitochondrial sites, such as peroxisomes, the H₂O₂ produced byMnSOD is converted into H₂O by catalase (CAT). Thus CAT functions in ROSdetoxification by protecting cells from the H₂O₂ that is generatedwithin them. In the present example, a significant decrease in CAT mRNAexpression in unfractioned biopsy RNA was observed in the secretoryphase of the Endo group compared to Controls (p=0.001), as shown inTable 3. Using a cut-off value of 0.2, the specificity and sensitivityof CAT is 83% and 78%, respectively (Table 5).

The glutathione S-transferase (GST) gene superfamily is one of the majordetoxifying and free-radical scavenging systems in mammalian cells. Inthe present example, a significant diminution in GST expression in theEXP 1 group compared with controls (p=0.026) was observed, asillustrated in Table 5. A direct analysis of Table 3 also shows anincrease in GST expression in the EXP 2 group. According to a cut-offvalue of 0.21, parameters for the EXP 1 group would be a specificity of54% and a sensitivity of 74% (Table 5).

Endothelial nitric oxide synthase (eNOS) catalyzes the oxidation ofL-arginine to nitric oxide (NO) and citrulline. Previous reports havedescribed an up-regulation at the protein level in the endometrium ofendometriosis patients during the secretory phase of the cycle (Ota etal., Fertility and Sterility (1998) 69:303-308). In the present example,a significant decrease in eNOS expression when comparing theproliferative phases of the control group and the proliferative phasesof the Endo group as a whole (p=0.027) is observed (see Table 3). Usinga cut-off value of 0.1, one could define specificity and sensitivity of77% and 65%, respectively (Table 5).

The so-called Cytochrome oxidase 3 (CO3) gene was published as beingup-regulated in hepatocytes by H₂O₂ and/or homocysteine. The sequenceamplified in the present example corresponded to several cDNAs inGENBANK™ such as the hypoxia-inducible gene 14 which was in fact in the16S rRNA region of the human mitochondrial genome, and with the TI-227HcDNA, a metastatic marker. Finally, the CO3 gene sequence is entirelyhomologous to the DD11 sequence (isolated by differential display). Theanalysis of the CO3 gene expression (and of DD11) revealed a secretoryphase-specific diminution, as seen in Table 3, when comparing theControl and Endo groups (p=0.002). When using a cut-off value of 0.58,the specificity and sensitivity of this marker were 83% and 79%,respectively (Table 5).

12S rRNA is one of the two rRNA genes encoded by the mitochondrialgenome. In the present example, a significant decrease in 12S rRNA inunfractioned biopsy RNA was observed in the EXP 1 group compared toControls (p=0.02), as illustrated in Table 3. Using a cut-off value of1, the specificity and sensitivity of 12S rRNA are 77% and 67%,respectively (Table 5).

TI-227H is a nuclear gene that has homologies with CO3, as describedabove. As a result of these sequence similarities, the present inventorswere interested in determining if the two sequences were, in fact, thesame gene, or were perhaps pseudogenes. Interestingly, analysis ofTI-227H mRNA expression in unfractioned biopsy RNA, described in thepresent example, showed different results from those obtained with CO3,suggesting that the two sequences are from different genes. Unlike CO3expression which decreased in the entire Endo group, expression ofTI-227H diminished significantly in the EXP 2 group compared to Controls(p=2×10⁻⁸), as shown in Table 3. The specificity and sensitivity ofTI-227H are 81% and 100%, respectively, when using a cut-off value of0.15 (Table 5).

Cytochrome oxidase 2 (CO2) is a mitochondrial-encoded enzyme thatparticipates in the mitochondrial electron-transport chain (ETC) andthus, in the process of oxidative phosphorylation (OXPHOS) whichprovides essential energy to cells. As shown in Table 3, CO2 mRNAexpression in unfractioned biopsy RNA was found to decrease in theproliferative phase of EXP 1 compared to Controls (p=0.02). When using acut-off value of 0.2, the specificity and sensitivity of CO2 are 64% and69%, respectively (Table 5). There is experimental evidence reported inthe literature showing changes in iron metabolism in response tooxidative stress. One of the key enzymes in this pathway is themitochondria-located aconitase.

Analysis of the aconitase gene expression (Table 3) showed a decrease inthe S-phase of EXP 1 group (p=0.005) and also in the P-phase of EXP 2(p=0.04). Analysis of mean values of P-phase samples exhibits a decreaseof the P-phase of both groups (EXP 1 and EXP 2), but only the latter issignificantly lower than the control group. Since the most remarkabledecrease is observed in the EXP 1 S-phase, we evaluated the diagnosticpower of aconitase on this basis. With a 0.11 cut-off, a specificity of80% and a sensitivity of 86% were calculated (Table 5).

Encoded by a nuclear gene, adenine nucleotide translocator 1 (ANT-1) islocated within the mitochondrial inner membrane. ANT-1 plays animportant role in OXPHOS, by exchanging newly-synthesized ATP for spentADP. To date, modulation of ANT-1 expression in endometrial biopsies hasnot been reported. In the present example, ANT-1 mRNA expressionanalyzed by RT-PCR in unfractioned biopsy RNA was found to decrease inthe proliferative phase of the Endo group compared to Controls(p=0.007), as shown in Table 3. When using a cut-off value of 1, ANT-1has a specificity of 86% and a sensitivity of 74% (Table 5). This resultwas confirmed by Northern-blotting (p=0.016) whereby the specificity andsensitivity values were 75% and 73%, respectively, when a cut-off valueof 0.042 was used (Table 5).

ATP synthase, also known as Complex V of the ETC, is encoded by twomitochondrial genes (ATPase 6 and ATPase 8) and 12 nuclear genes. Inagreement with its critical role in OXPHOS, mutations in ATP synthasehave been associated with mitochondrial diseases. As shown in Table 3,the expression of the β subunit of ATP synthase in unfractioned biopsyRNA increased in the Endo group compared to Controls (p=1×10⁻³).Interestingly, when considering only the proliferative phase of the Endogroup, the statistical analysis becomes p=8×10⁻⁴. When using a cut-offvalue of 4, the specificity and sensitivity of ATP synthase (β subunit)is 77% and 75%, respectively, when comparing the Endo group andControls. When analyzing the P-phase of the Endo group, the specificityis 93% and the sensitivity is 64% (Table 4).

B-cell leukaemia/lymphoma-2 (Bcl-2) is a nuclear-encoded anti-apoptoticprotein that is localized in the mitochondrial outer membrane.Interestingly, Bcl-2 expression seems to be driven by estradiol in theendometrium throughout the ovarian cycle, whereby high Bcl-2 expressionis observed in the proliferative endometrium and is decreased in thesecretory phase (Vaskivuo et al (2000) Mol Cell Endocrinol 165:75-83).Previous studies have analyzed Bcl-2 expression in glandular versusstromal cells, and in eutopic versus ectopic endometrial fractions(Meresman et al. (2000) Fertil. Steril. 74(4): 760-6; Jones et al.(1998) Hum. Reprod. 13(12): 3496-502). In the present study, Bcl-2expression in unfractioned endometrial biospsy RNA decreased in the Endogroup compared to Controls (p=0.01), as shown in Table 3. Thespecificity and sensitivity of Bcl-2 expression are 54% and 82%,respectively, when using a cut-off value of 4 (Table 5).

Human c-jun proto oncogene (c-jun) is one of the major ubiquitoustranscriptional factors which forms the AP1 complex with c-fos. In thepresent example, c-jun expression was analyzed and a significantup-regulation during the secretory phase of the EXP 1 group (p=0.05) wasobserved (see Table 3). With a cut-off value of 0.7, the followingparameters for the EXP 1 S-phase group were obtained: specificity 75%,sensitivity 67% (Table 4). When tested on unfractioned biopsy RNA,up-regulation of c-jun expression was significant for the Endo groupcompared to the Control group (p=0.005). With a cut-off value at 1, aspecificity of 76% and a sensitivity of 65% were determined (Table 4).

Chicken ovalbumin upstream promoter transcription factor (COUP-TF) is animportant regulator of aromatase P450 (P450arom), which is involved inestrogen biosynthesis. In eutopic endometrial stromal cells, binding ofCOUP-TF to a region upstream of the aromatase promoter mediates theinhibition of aromatase transcription, thus preventing aberrant estrogenproduction. As shown in Table 3, COUP-TF mRNA expression decreased inthe secretory phase of the Endo samples compared to Controls (p=0.01) inunfractioned RNA biopsies. Using a cut-off value of 0.075, thespecificity and sensitivity of COUP-TF is 83% and 78%, respectively(Table 5).

Interleukins are involved in the inflammatory process, as encountered indiseases like endometriosis. Transcription analysis of IL-1β in thepresent example revealed a totally impaired pattern of expression in theEndo group, while the level is rather constant in the control group (seeTable 3). In particular, a significant decrease in IL-1β expression wasobserved in the EXP 2 group (p=0.0002) Based on a scatter graph (notshown) two cut off values were determined, one for a mean value for theControl group and the other for its corresponding standard deviation.Values outside of this range were identified as corresponding topatients with a high likelihood of suffering from endometriosis. Aspecificity of 78% and a sensitivity of 93% for the Control group meanvalue of 0.98±0.28 were determined (Table 5).

Connexins are involved in cell interactions and, more specifically, instructures such as gap junctions. Their expression is generallyregulated by steroids. Connexin 43 (Cx43) seems to be regulated byestradiol. Generally in the endometrium, connexin 43, as well as someother connexins, show a higher expression by immunohistochemistry ataround 11-15 days of the menstrual cycle. They are thought to bespecifically involved in implantation, for example during pregnancy. Onepublication reported an aberrant expression of connexin 43 in allectopic epithelial tissue samples in endometriotic lesions. No studieshave ever measured the level of expression of connexin 43 in eutopicendometrial cells. In the present example, an up-regulation of Cx43 mRNAwas observed in the EXP 1 group compared to controls (p=0.048) (Table3), a difference which was even more significant when only theproliferative phase of the EXP 1 group was considered (p=0.02). With acut-off value of 1.3, a specificity of 74% and a sensitivity of 48% forthe EXP 1 group were obtained (Table 4). By considering theproliferative phase of the EXP 1 group, the marker is more powerful,since the specificity and the sensitivity are 77% and 64%, respectively(Table 4). Heat shock proteins (HSPs) are often induced to protect cellsfrom various stresses. More specifically, heat-shock protein 70 (HSP 70)is induced in response to protein misfolding, DNA damage, metabolicoxidative stress, and hypoxia. It is reported in the present examplethat the modulation of HSP 70 mRNA in women with endometriosis issharply elevated in the Endo group compared to the Control group(p=0.01) (see Table 3). As a marker for endometriosis, the specificityand sensitivity of HSP 70 were 73% and 61%, respectively, when using acut-off value of 0.6 (Table 4). On unfractioned biopsy RNA, asignificant increase in the proliferative phase was also detected(p=0.014), the marker thus having a specificity of 71% and a sensitivityof 78% with a cut-off at 0.25 (Table 4).

Heat-shock protein 90 (HSP 90) is part of a protein chaperone systeminvolved in steroid-inducible and exogenous toxin-inducible signaling.In the present example, the analysis of HSP 90 in regard toendometriosis revealed a marked decrease in gene expression whencomparing women in the EXP 2 group with Controls (p=4.3×10⁻⁵) (see Table3). When a cut-off value of 0.17 was used in the analysis of the EXP 2group compared to the control group, the specificity and sensitivitywere 75% and 100%, respectively (Table 5).

Phospholipid hydroperoxide glutathione peroxidase 4 (GPx4 or PHGPx) isresponsible for reducing hydroperoxides that are produced in peroxidizedmembranes and oxidized lipoproteins. In unfractioned biopsy RNA, shownin Table 3, GPx4 mRNA expression decreased in the secretory phase of theEXP I group compared to Controls (p=5×10⁻³). Using a cut-off of 0.1, thespecificity of GPx4 is 83% and the sensitivity is 75% (Table 5).

Glucose-regulated protein 78 (GRP 78) is a stress-response proteinrelated to HSP70 that is induced by agents or conditions that adverselyaffect the function of the endoplasmic reticulum, such as homocysteine.In the present example, expression of GRP 78 in unfractioned biopsy RNAdecreased in the secretory phase of the Endo group compared to Controls(p=6×10³), as shown in Table 3. The specificity and sensitivity of GRP78 are 92% and 78%, respectively, when using a cut-off value of 3.

Cyclooxygenase-2 (cox2), also called prostaglandin synthase-2 (PG-2), isinvolved in prostaglandin synthesis. Its possible involvement inendometriosis has been discussed in relation to increased aromataseactivity in ectopic lesions but, to date, there is no evidence of cox2dysregulation in eutopic endometrial cells. In unfractioned biopsy RNA,shown in Table 3, cox2 mRNA expression significantly increased in theEXP 1 group compared to Controls (p=0.01). Using a cut-off value of 6,the specificity of COX2 is 69% and the sensitivity is 62% (Table 4).

III—Combination of Markers

Having found many genetic markers differentially expressed in Controland Endo groups, the inventors tested the hypothesis that differentiallyexpressed genes used in combination might provide a more powerfuldiagnostic tool than evaluation of single genes. This is especially truegiven the high level of genetic heterogeneity between individuals.

Table 11 show examples of combination of genetic markers. Specificityand sensitivity were calculated as described above (see statisticalanalysis). Briefly, specificity indicates the absence of a marker in thecontrol group and sensitivity indicates the presence of a marker in theENDO group. All 3 genetic markers of interest were tested on the sameindividual patient samples, thus allowing combinations to be made. Foreach patient sample, the presence of a given marker was assigned a scoreof 1, while the absence of the same marker was assigned a score of 0(the presence and absence of a marker are defined according to beingabove or below the assigned cut-off value). Following the application ofthis algorithm to all 3 genes of interest, the marker scores werecombined and converted to percentages according to the following: forcombinations of 2 markers, 0% represents a score of 0 for both markers,50% represents a score of 0 for 1 marker and a score of 1 for the othermarker, and 100% represents a score of 1 for both markers; forcombinations of 3 markers, 0% represents a score of 0 for all 3 markers,33% represents a score of 1 for ⅓ markers, 67% represents a score of 1for ⅔ markers, and 100% represents a score of 1 for all 3 markers. Thesepercentages were then graphed, and new specificity and sensitivityvalues were calculated—these values are indicated in Table 11.

An example of the power of combinations of markers is shown in Table 11,whereby a combination of two markers increased the sensitivity of thediagnosis up to 100%. A combination of three markers increased bothspecificity and sensitivity. Other combinations of numerous markerswould also yield improved specificity and sensitivity, therebyameliorating the methods and kit according to the present invention.

4) Conclusion

The results presented herein show that the genetic makers listed inTable 1 are all excellent endometriosis markers. The use ofdifferentially expressed genes therefore represents an alternative meansto identify diseased individuals and grade endometriosis. By combiningtwo, three and more of these markers, it is also possible to increasethe specificity and the sensitivity of the diagnosis.

Many of the genes among the markers of the present invention areassociated with mitochondria. Five among the sixteen DD markers were inthis category. This observation led the inventors to further investigatein this field. Indeed, physiologically a number of key metabolicreactions, such as oxidative phosphorylation (OXPHOS) imply themitochondria. Hence, as shown in Table 3, modulation of 22 OXPHOS-and/or mitochondria-related genes, such as HIF-1α, ARNT or NADHdehydrogenase, was observed in the endometrial tissue of endometriosispatients. This strongly suggests that the OXPHOS pathway is involved inendometriosis and that it is possible to determine the likelihood ofendometriosis in a female subject by assaying endometrial cells samplefor the expression level of at least one endometriosis-related markerinvolved in the oxidative phosphorylation pathway and/or the internalredox potential sensors pathway (OXPHOS) of said endometrial cells.

Interestingly, three differentially expressed cDNAs containing alusequences were isolated and identified as endometriosis-related markers.Similarly, another heterogeneous group of endometriosis-related markerswere cellular stress markers (heat shock proteins, CAP43, Connexin 43,RNA helicase). It is therefore tempting to speculate that most genescontaining an alu sequence and/or cellular stress markers could be usedas endometriosis-related markers according to the present invention.

Example 2 Proteinic Markers

1) Problematic

Having found many genetic markers differentially expressed in Controland Endo groups of women, the inventors tested the hypothesis thatprotein measurement of protein product(s) derived from these geneticmarkers could also be a suitable approach for the diagnosis ofendometriosis.

Since the marker DD5 (namely Cap43) was deregulated at the mRNA level inthe endometrium of women suffering from endometriosis, the presentinventors have characterized the expression of the protein encoded bythe same gene. This was performed by Western blotting.

Cap43 mRNA has a 1759 bp 3′-untranslated region and its predicted openreading frame encodes a 394 amino acid residue polyprotein with adeduced molecular weight of 43,400 Dalton and an isoelectric point of5.3. Previous studies have shown a modulated expression of the Cap43gene's mRNA in various types of cells. However, the Cap43 protein hasnever been used as a marker in any disease, including endometriosis. Inthe present example, protein extracts isolated from endometrial tissuebiopsies from control and diseased populations were screened for Cap43protein expression and the results presented herein show that thisprotein is a suitable marker for endometriosis

2) Materials and Methods

Patient Recrutment and Tissue Samples

Tissue biopsies from control and endometriosis-positive populations wereobtained as described previously in Example 1 herein.

Isolation and Preparation of Proteins from Unfractioned Biopsies

Total cellular protein extracts were isolated directly from tissuebiopsies. Briefly, 200-300 mg of biopsy tissue was homogenized using anelectric homogenizer in the presence of 3 ml of lysis buffer (20 mMTris-HCl pH 7.5 containing 0.1 M NaCl, 2% SDS, 5 mM EDTA and 0.5 μg/mlleupeptin, 2 μg/ml aprotinin and 200 μg/ml PMSF) at room temperature,lysed 10 min at 100° C. and centrifuged 20 min at 14000 g. Proteindetermination of supernatants was made using the DC protein assay(BIO-RAD™) according to the supplier's instructions. Samples werediluted in 20 mM Tris-HCl pH 7.5 and dotted onto nitrocellulosemembranes (2.5 and 5 μg) using a 96-wells GIBCO-BRL™ dotter device. Themembranes were blocked overnight in 2.5% dry milk powder in TST buffer(10 mM tris-HCl pH 7.5 containing 100 mM NaCl and 0.1% TWEEN-20™),incubated in a dilution of either 1:2000 of primary antiserum anti-CAP43 (SKULDTECH™, Université Montpelier II, France) or anti-actin (SANTACRUZ BIOTECHNOLOGY™) for one hour, washed twice for 5 min with TST andincubated for another hour with an horseradish-peroxidase(HRP)-conjugated goat anti-rabbit immunoglobulin-specific polyclonalantibody (1:2000 dilution in 5% milk-TST), washed 3 times 10 min withTST and visualized by ECL reagent according to the supplier's protocol(AMERSHAM PHARMACIA BIOTECH™). Levels of Cap43 relative to actin weremeasured by scanning the autoradiograms and densitometry of dots wasperformed using the program MOLECULAR ANALYST™.

3) Results

Using anti-CAP 43 polyclonal antibodies, the present inventors screenedprotein extracts isolated from endometrial tissue biopsies from controland diseased populations. As shown in Table 3, two types of experimentswere performed. In the first experiment, all proliferative and secretorycontrol and experimental groups were included in order to compare thepresence of Cap43 in both phases of the ovarian cycle. In the secondexperiment, only secretory phase groups were tested, and in this way,the number of samples analyzed could be increased.

Results from the first experiment show that the Cap43 protein wasexpressed in higher levels in the secretory phase of the ovarian cycle,with a mean upregulation in the control group of more than two fold inthe secretory phase compared to the proliferative phase (1.92±0.21 CTL Svs. 0.55±0.06 CTL P). In addition, by comparing the control and Endogroups, it appears that there is a significant decrease in Cap43 in thesecretory phase of the Endo group (p=0.05), with specificity andsensitivity values of 71% and 57%, respectively, when a cut-off of 1.35is used (Table 5).

In the second experiment in which only samples in the secretory phasewere analyzed, there is also a significant decrease in the secretoryphase of the Endo group (p=0.001). Using a cut-off of 1.1, thespecificity is 77% and the sensitivity is 61% (Table 5).

4) Conclusion

These results confirm that, like the Cap43 mRNA, the Cap43 protein isdifferentially expressed in the endometrial cells of endometriosis-freecompared to women having endometriosis. It is therefore justified tobelieve that it is the same for all the endometriosis-related geneticmarkers listed in Table 1.

The use of differentially expressed genes or their derived proteinproducts (such as translated proteins or peptides) therefore representsan alternative means for determining the likelihood of endometriosis infemales and for grading this disease in female suffering from it.

TABLE 1 Endometriosis-related markers GENBANK ™ Reference ApplicantReference No of SEQ ID NO Arbitrary name GENBANK ™ gene name AccessionNo Type bases Date 1 DD1 — AL050039; AC007064 EST; EST 6241 18-Feb-00124823 27-May-00 2 DD2 — AF084555 EST 5171 1-Sep-99 3 DD3 — J01415 DNA16569 18-Apr-00 4 DD4 — AA829538 EST 1329 29-Apr-98 5 DD5Nickel-specific induction protein (CAP43) AF004162 mRNA 2972 24-Jan-00 6DD6 — AL034374 EST 101270 29-Apr-00 7 DD7 — AI567884; EST; 500 14-May-99AA558871; EST; 487 9-Sep-97 AI890794 EST 2501 7-Mar-00 8 DD8 RNAhelicase AB028449 mRNA 7037 18-Feb-00 9 DD9 NADH dehydrogenase AF004342DNA 320 19-Jul-97 J01415 DNA 16569 18-Apr-00 10 DD10 NADH dehydrogenaseAF014897 DNA 1041 6-May-99 J01415 DNA 16569 18-Apr-00 11 DD11 CytochromeOxidase 3 (CO3) AB017708 DNA 346 26-Sep-98 12 DD12 transcription factorForkhead domain protein (FKHR) AF032885 mRNA 5723 19-Feb-98 13 DD13hUCC1 AJ250475 mRNA 2073 1-Jul-00 14 DD14 Paralemmin AK000278 mRNA 219722-Feb-00 15 DD15 Citrate transport protein X96924 DNA 2270 9-Oct-97 16DD16 — AC022148 DNA 198751 26-Aug-00 12S mitochondrial RNA (12S rRNA)J01415 DNA 16569 18-Apr-00 Hypoxia-induced factor 1α (HIF1α) NM_001530mRNA 3933 31-Oct-00 Aryl hydrocarbon Receptor Nuclear Translocator(ARNT) NM_001668 mRNA 2616 31-Oct-00 Adenylate Kinase isozyme 3 (AK3)X60673 mRNA 1707 18-Jan-95 Glucose Transporter isoform 1 (Glut-1)aa368897 mRNA 288 21-Apr-97 Manganese SuperOxide Dismutase (MnSOD)X14322 mRNA 977 12-Nov-90 Glutathione Peroxidase (GPx) X13709 mRNA 8196-Apr-95 Catalase (CAT) NM_001752 mRNA 2279 31-Oct-00 GlutathioneS-transferase (GST) X15480 mRNA 725 12-Sep-93 Endothelial nitric oxidesynthase (eNOS) M93718 mRNA 4077 27-Apr-93 TI227H DD50525 mRNA 391110-Feb-99 Cytochrome Oxidase 2 (CO2) J01415 DNA 16569 18-Apr-00aconitase NM_001098 mRNA 2467 31-Oct-00 Adenine Nucleotide Translocator1 (ANT-1) NM_001151 mRNA 1320 31-Oct-00 ATP synthase X03559 mRNA 180730-Dec-97 B-cell leukaemia/lymphoma-2 (Bcl-2) NM_000633 mRNA 60303-Feb-01 Human c-jun proto oncogene (c-jun) J04111 DNA 3622 6-Jan-95Chicken Ovalbumin Upstream Promoter Transcription X16155 mRNA 151319-Jul-95 Factor (COUP-TF) Interleukin 1β (IL-1β) m15330 mRNA 14976-Jan-95 Connexin 43 (Cx43) m65188 mRNA 1314 1-Nov-94 Heat-Shock Protein70 (HSP 70) m11717 DNA 2691 8-Nov-94 Heat-Shock Protein 90 (HSP 90)x15183 mRNA 2912 30-Jan-95 Phospholipid hydroperoxide glutathioneperoxidase 4 NM_002085 mRNA 896 31-Oct-00 (GPx4) Glucose-regulatedprotein 78 (GRP78) m19645 DNA 5470 8-Nov-94 Cyclooxygenase-2 (cox2)m90100 mRNA 3387 31-Dec-94

TABLE 2 Nucleotide sequences of endometriosis-related markersidentified by differential display SEQ  Arbitrary ID NO NameNucleotide Sequence 1 DD1ggttagtaattctgcagatcgctagctcgacgattcattggctgaatagccagtggtgcaggacatatgcacagtgtctgacctcagtaacttcactctcatacatatgtattaggacaccaacacatgtgtgcatataagatgtatgatagatattgcaacaagtaataatttactgtcctatttataggattttaaacttaaactactttcaccctatttccaaaaaaa 2 DD2gactgtactgaaagggccaagagtaaatgccttcgttttgtttttttcgtttnttttgttttagctttttgttaaaacgtctatagattggcagttaatgctgaatttgtcaaataccccttccaaaattatactttgtatttaaaaaataaatgggatctacctaatttccaa 3 DD3cgactgtatgntgaacgtaggtgcgataaataataggatcgaggcaggaatcaaagacagatactgcgacatagggtgctccggctccagcgtctcgcaatgctatcgcgtgcatacccccaacgactgtggacgagagg 4DD4gaacctggtggtgggaccatggaggcagggtgcagaggtgcacaataaaattgattatcatcgtttttgagaatgttgttggtttccccca 5 DD5aagctttggtcagagtgaattgaatattgtaagtcagccactgggacccgaggattctgggaccccgcagttgggaggaggaatnagtccagccttccaggtggcgtgagaggcaatgactcgttacctgccgcccatcaccttggaggccttccctggccttgagtagaaaagtcggggatcggggcaagagaggctgagtacggatgggaaactattgtgcacaagtctttccanaggagtttcttaatgagagatttgtatttatttccagaccaataaatttgtaactttgcaa 6 DD6aagctttggtcagggatagagaatgaaagtgagatcatttagatcttagaaaggnagatgttnggctngggcacggtggctcacacctgtaatcccagcacttgggaagccatggtgggcagatcatttgagctcaggagtttgcaaccagcctgggcaatatggcaagaccccatctgtacaa 7 DD7ttgtatttttagtaaagacggggtttcactatgttggccaggctggtctcgaactcctgacctcgtgatccacccaccttggcctcccaatcttatttgctttacaagtcctgcttcagggttaccttccctgaccaaagctt 8 DD8tctaatgcataataaaatgaaaggaatcgtaaaacagtttcgttccaaaaagtcagagataaagactatccatgaaggttcacttttgaggcaagaacccttttttatgcaagactatgtggcatcagaaaactaaaatgtgattcaccaacatgccagccaatgttcattaaaaatctgtcccttactaacaggtgcaacagcgaccgggaacatcaccttacacagtataacgtggaaagaaaagacaacattgggngcacttctcntctccaaaaccttatctttcnattcagctttancatntactgcaggactg 9 DD9ttgattcggttcagtctaatcctttttgtatcactcataggccagacttnagggctaggatgatgattaataagagggatgacataactattagtggcaggtagttgtttgtagggctcatggtaggggtaaaaggagggcaatttctagatcaaataataagaaggtaatagctactaagaagaattttatggagaaagggacgcgggcgggggatatagggtcgaagccgcactcgtaaggggtggatttttctatgtagccgttgaagaagctt 10DD10gtaggcagttgaggtggattaaaccaaacccagctacgcaaaatcctnagcatactcctcaattacccacataggatgaataatagcagttctaccgtacaaccctaacataacctgcttaatttaactatttatattatcctaactactaccgcattcctactactcaacttaaactccagcaccacgaccctactactatctcgcacctgtaacaagctaacatgactaacacccttaattccatccaccctcctctccctaggaggcctgaccccgctaancgngctttttgcccaattgggcattancgagattca 11 DD11gtaggcctaaaagcagccaccaattaagaaagcgttcaagctcaacacccactacctnaaaaaatcccaaacatataactgaactcctcacacccaattgngaccaatctatcaccctatagaagaactaatgttagtataagtaacatgaaaacattctcctccgcataagccttgcgtcagattaaaacactgaactgacaattaacagcccaatatctacaatcaaccaacaagtcattattaccctcactgtcaacccaacacaggcatgctcataaggaaaggttaaaaaaagtaaaaggaactcggcaaatcttaccncgc 12 DD12gactgtgacatggaatccatcattcggaatgacctcatggatggagatacattggattttaactttgacaatgtgttgcccaaccaaagcttcccacacagtgtcaagacaacgacacatagctgggtgtcaggctgagggttagtgagcaggttacacttaaaagtacttcagattgtctgacagcaggaactgagagaagcagtccaaagatgtctttcaccaactcccttttagttttcttggttaaaaaa 13 DD13ggttgagtttgtccattgctagggagagacttccagtaataaaatttactattctagatgcttctactgttatgttttatctacccatttatctttcttagttaccaggagaaatgtgtgacacctatattataatgaaaacaatcttattacttatagtttatctatattaaacaaatttaattgcatttaaagcattctttgatattgttgcttttgcaataaatatggataatcttggttataagggagttaaaacaatgctgtaataaataaagtgtttcatgtgatcaaa 14 DD14ttcatcatcttctttttcctcatnnatctccttccttaacctagaaggtatgtaggactttggaaggtcagggatattagcatagatgtcctcaattgactcttctgctctttcttctctttccactttcacagatcttataatgtcttctgttgtccgctcaattgactttagtttctttaaaatggcctcttccttcgttgagaagcttaagccga 15 DD15ggcctggcttcaccgcattccaggctgcagccccctgcttctcccgccattgccttaactgccctcgggccctctctccgccccggacagggtggcacccaccactctcaggaccaccctgccaaggcagaataaaccggatcctgttgc 16 DD16aagcttgcaccatgacctaacgttttatgtaaatacttgtgtttagtaccttttaaggttttgcagaagatggcggtgtataggctgaattagcaagagatagtgaggtttactggggtttattgattcaaa

TABLE 3 Summary of average values (± SEM, standard error of the mean) bygroup for the endometriosis-related markers Name CTL P

CTL

CTL EXP I P EXP I S EXP I EXP II P EXP II S EXP II ENDO P ENDO S ENDODD1 (epith) 0.24 ± 0.04 0.81* ± 0.20  0.55 ± 0.12 0.60 ± 0.21 0.15* ±0.03  0.38 ± 0.12 2.77 ± 1.96 1.39 ± 1.02 1.94 ± 0.97 1.36 ± 0.72 0.74 ±0.49 1.03 ± 0.42 DD2 (epith) 1.21 ± 0.62 0.42 ± 0.14 0.76* ± 0.29  0.10± 0.04 0.09 ± 0.03 0.10 ± 0.02 0.09 ± 0.02 0.28 ± 0.13 0.21 ± 0.08 0.10± 0.03 0.18 ± 0.06 0.14* ± 0.03  DD3 (epith) 0.98 ± 0.09 0.64 ± 0.030.88* ± 0.07  0.79 ± 0.09 0.57 ± 0.04 0.68 ± 0.05 0.41 ± 0.10 0.60 0.45± 0.08 0.71 ± 0.08 0.58 ± 0.04 0.65* ± 0.05  n.a. DD4 (epith) 0.87 ±0.18 1.29* ± 0.23  1.00 ± 0.15 0.77 ± 0.12 0.79 ± 0.14 0.78 ± 0.09 0.80± 0.36 0.36 0.69 ± 0.28 0.78 ± 0.11 0.75* ± 0.13  0.77 ± 0.08 n.a. DD5(epith) 1.92* ± 0.38  3.65 ± 1.85 2.86 ± 1.02 0.85 ± 0.25 1.61 ± 0.501.19 ± 0.27 1.11 ± 0.62 28.06 ± 23.75 17.70 ± 14.73 0.92* ± 0.24  13.37± 10.65 7.32 ± 5.51 DD5 n.a. 1.81* ± 0.17  n.a. n.a. 1.08 ± 0.11 n.a.n.a. 1.34 ± 0.29 n.a. n.a. 1.12* ± 0.10  n.a. (biop 0.55 ± 0.06 1.92* ±0.21  1.28 ± 0.18 0.62 ± 0.07 1.45 ± 0.13 1.05 ± 0.11 n.a. 0.93 ± n.a.n.a. n.a. 1.41* ± 0.12  1.04 ± 0.11 PROT⁺) (2 experiments) DD6 (epith)2.18 ± 1.32 0.83* ± 0.23  1.42 ± 0.59 0.49 ± 0.12 0.15* ± 0.03  0.32 ±0.08 1.06 ± 0.72 0.54 ± 0.20 0.74 ± 0.29 0.73 ± 0.30 0.36 ± 0.12 0.52 ±0.15 DD7 (epith) 1.49* ± 0.39  2.47 ± 1.37 2.02 ± 0.76 0.38 ± 0.12 0.35± 0.09 0.37 ± 0.08 0.27 ± 0.21 0.99 ± 0.34 0.73 ± 0.24 0.35* ± 0.10 0.65 ± 0.18 0.51 ± 0.11 DD8 (epith) 0.81 ± 0.14 1.01 ± 0.20 0.88* ±0.11  0.59 ± 0.10 0.52 ± 0.12 0.56* ± 0.08  0.70 ± 0.22 0.73 ± n.a. 0.70± 0.17 0.62 ± 0.09 0.54 ± 0.11 0.58 ± 0.07 DD9 (epith) 0.14* ± 0.03 0.41 ± 0.14 0.29 ± 0.08 0.29 ± 0.06 0.44 ± 0.22 0.36 ± 0.10 0.76 ± 0.230.35 ± 0.06 0.52 ± 0.11 0.43* ± 0.09  0.40 ± 0.13 0.42 ± 0.08 DD10(epith) 0.17* ± 0.03  0.23 ± 0.02 0.19 ± 0.02 0.27* ± 0.04  0.13 ± 0.020.20 ± 0.03 0.21 ± 0.06 0.08 0.18 ± 0.05 0.26 ± 0.03 0.13 ± 0.02 0.20 ±0.02 n.a. DD11 (biop) See CO3 DD12 27.92 ± 5.01  40.20 ± 5.46  34.73* ±3.97  14.48 ± 2.29  35.42 ± 3.85  26.11 ± 3.42  17.10 ± 42.96 ± 5.10 20.79 ± 3.04  16.05 ± 1.46  36.67 ± 3.35  23.78* ± 2.35  (biop) 1.91DD12 0.47 ± 0.14 1.15 ± 0.26 0.78* ± 0.15  0.46 ± 0.15 0.52 ± 0.15 0.49± 0.10 0.27 ± 0.13 0.53 ± 0.33 0.31 ± 0.12 0.37 ± 0.10 0.52 ± 0.13 0.42*± 0.09  (biop NB) DD13 (biop) 0.72 ± 0.13 0.24 ± 0.08 0.45* ± 0.09  0.83± 0.17 0.24 ± 0.06 0.48 ± 0.10 0.87 ± 0.17 0.47 ± 0.11 0.82* ± 0.15 0.86 ± 0.12 0.28 ± 0.06 0.63 ± 0.09 DD14 (biop) 1.36 ± 0.25 0.77 ± 0.121.03* ± 0.15  2.65 ± 0.83 1.23 ± 0.18 1.86 ± 0.41 1.18 ± 0.15 2.06 ±0.13 1.31 ± 0.15 1.77 ± 0.37 1.37 ± 0.18 1.62* ± 0.24  DD15 (biop) 0.12± 0.07 0.10 ± 0.06 0.11* ± 0.05  0.49 ± 0.12 0.32 ± 0.08 0.43 ± 0.080.48 ± 0.15 0.12 ± 0.12 0.39 ± 0.13 0.49 ± 0.09 0.28 ± 0.07 0.42* ±0.07  DD16 (biop) 2.54 ± 0.35 2.46 ± 0.32 2.50* ± 0.23  0.63 ± 0.16 1.49± 0.11 0.98 ± 0.15 1.63 ± 0.33 1.90 ± 0.19 1.70 ± 0.24 0.96 ± 0.19 1.58± 0.11 1.19* ± 0.14  HIF1α 0.84 ± 0.10 1.34 ± 0.15 0.99* ± 0.10  1.29 ±0.13 1.23 ± 0.16 1.26 ± 0.10 1.47 ± 0.33 2.27 1.62 ± 0.30 1.33 ± 0.121.31 ± 0.17 1.32* ± 0.10  (epith) n.a. HIF1α 0.78* ± 0.08  1.82 ± 0.351.20 ± 0.18 1.06 ± 0.14 1.82 ± 0.32 1.35 ± 0.18 1.16 ± 0.14 1.27 ± 0.711.19 ± 0.21 1.11* ± 0.10  1.61 ± 0.32 1.28 ± 0.13 (biop NB) ARNT 0.53* ±0.11  1.38 ± 0.65 1.00 ± 0.37 1.04 ± 0.19 1.76 ± 0.61 1.37 ± 0.30 1.03 ±0.17 10.13 ± 7.93 6.49 ± 4.80 1.04* ± 0.14  5.72 ± 3.78 3.44 ± 1.96(epith) AK3 (epith) 0.60 ± 0.17 1.62 ± 0.32 1.14* ± 0.22  0.22 ± 0.070.62 ± 0.18 0.41* ± 0.10  3.16 ± 2.12 13.83 ± 10.70 8.91 ± 5.83 1.40 ±0.89 6.78 ± 5.10 4.10 ± 2.59 Glut-1 0.51 ± 0.12 0.86 ± 0.18 0.69* ±0.11  0.63 ± 0.21 1.28 ± 0.65 0.81 ± 0.29 1.23 ± 0.39 2.33 ± 0.77 1.87*± 0.49  0.81 ± 0.19 1.77 ± 0.50 1.26 ± 0.27 (epith) MnSOD 0.62 ± 0.040.96 ± 0.23 0.84* ± 0.15  0.10 ± 0.23 3.89 ± 2.32 2.62 ± 1.32 5.35 ±4.12 6.29 ± 2.85 5.90 ± 2.27 2.81 ± 1.73 4.94 ± 1.77 4.03* ± 1.25 (epith) MnSOD 0.68 ± 0.06 0.89 ± 0.16 0.76* ± 0.08  0.92 ± 0.18 2.20 ±0.41 1.56 ± 0.26 0.89 ± 0.07 1.36 ± 0.30 0.98 ± 0.29 0.90 ± 0.09 2.00 ±0.33 1.30* ± 0.16  (biop) GPx (epith) 0.34 ± 0.08 0.80 ± 0.11 0.49* ±0.08  1.50 ± 0.18 2.01 ± 0.19 1.75 ± 0.14 1.83 ± 0.35 2.80 2.02 ± 0.331.57 ± 0.16 2.08 ± 0.18 1.80* ± 0.13  n.a. catalase 0.60 ± 0.08 0.49* ±0.08  0.55 ± 0.06 0.88 ± 0.25 0.13 ± 0.04 0.59 ± 0.17 0.46 ± 0.18 0.140.41 ± 0.16 0.74 ± 0.18 0.13* ± 0.03  0.55 ± 0.14 (biop) n.a. GST(epith) 0.40 ± 0.09 0.38 ± 0.12 0.39* ± 0.07  0.21 ± 0.08 0.15 ± 0.030.19* ± 0.05  0.80 ± 0.48 0.67 ± 0.21 0.71 ± 0.20 0.35 ± 0.13 0.42 ±0.13 0.39 ± 0.09 eNOS (epith) 0.35* ± 0.08  0.39 ± 0.20 0.36 ± 0.08 0.17± 0.05 0.40 ± 0.09 0.27 ± 0.05 0.07 ± 0.03 0.04 0.06 ± 0.02 0.15* ±0.04  0.37 ± 0.09 0.24 ± 0.05 n.a. CO3/DD11 0.44 ± 0.09 0.77* ± 0.110.55 ± 0.08 0.51 ± 0.08 0.31 ± 0.07 0.41 ± 0.06 0.35 ± 0.19 0.03 0.29 ±0.16 0.47 ± 0.07 0.29* ± 0.07  0.39 ± 0.05 (epith) n.a. 12S rRNA 4.12 ±1.02 3.02 ± 0.70 3.61* ± 0.63  0.78 ± 0.42 2.82 ± 1.19 1.56* ± 0.55 4.57 ± 1.83 1.68 4.16 ± 1.60 1.98 ± 0.74 2.69 ± 1.06 2.21 ± 0.60 (biop)n.a. TI227H 0.33 ± 0.04 0.30 ± 0.06 0.32* ± 0.04  0.43 ± 0.12 0.45 ±0.18 0.44 ± 0.10 0.02 ± 0.01 0.05 ± 0.05 0.03* ± 0.01  0.30 ± 0.09 0.37± 0.15 0.33 ± 0.08 (biop) CO2 (biop) 0.58* ± 0.16  0.37 ± 0.15 0.48 ±0.11 0.16* ± 0.04  0.40 ± 0.05 0.25 ± 0.04 0.83 ± 0.20 2.42 1.06 ± 0.280.37 ± 0.10 0.62 ± 0.23 0.45 ± 1.10 n.a. aconitase 0.43 ± 0.10 0.18* ±0.02  0.31 ± 0.06 0.30 ± 0.07 0.07* ± 0.02  0.21 ± 0.05 0.20 ± 0.03 0.30± 0.04 0.22 ± 0.03 0.25 ± 0.04 0.12 ± 0.04 0.21 ± 0.03 (biop) ANT-10.02* ± 0.003 0.03 ± 0.01 0.03 ± 0.004 0.01 ± 0.001 0.08 ± 0.02 0.03 ±0.01 0.02 ± 0.01 0.00 0.01 ± 0.005 0.01* ± 0.002 0.07 ± 0.02 0.03 ± 0.01(biop) n.a. ANT-1 0.09* ± 0.02  0.02 ± 0.01 0.06 ± 0.01 0.05 ± 0.02 0.02± 0.01 0.04 ± 0.01 0.02 ± 0.01 0.10 ± 0.06 0.05 ± 0.02 0.04* ± 0.01 0.05 ± 0.03 0.04 ± 0.01 (biop NB) ATP 2.28 ± 0.32 5.28 ± 1.53 3.67* ±0.77  5.06 ± 1.05 9.43 ± 1.43 6.72 ± 0.95 11.26 ± 2.19 13.14  11.53 ±1.87 7.02 ± 1.18 9.85 ± 1.33 7.93* ± 0.93  synthase n.a. (biop) 2.28* ±0.32  5.28 ± 1.53 3.67 ± 0.77 5.06 ± 1.05 9.43 ± 1.43 6.72 ± 0.95 11.26± 2.19 13.14  11.53 ± 1.87 7.02* ± 1.18  9.85 ± 1.33 7.93 ± 0.93 n.a.Bcl-2 (biop) 4.61 ± 1.13 6.11 ± 1.64 5.30* ± 0.97  3.96 ± 0.90 0.82 ±0.31 2.76 ± 0.65 0.99 ± 0.38 2.81 1.25 ± 0.41 3.02 ± 0.70 1.04 ± 0.352.38* ± 0.51  n.a. c-jun (epith) 0.51 ± 0.09 0.51* ± 0.08  0.51 ± 0.070.53 ± 0.10 0.85* ± 0.11  0.66 ± 0.08 0.63 ± 0.31 0.10 0.50 ± 0.26 0.55± 0.09 0.77 ± 0.12 0.64 ± 0.08 n.a. c-jun (biop) 0.68 ± 0.11 1.02 ± 0.180.82* ± 0.11  0.98 ± 0.26 1.84 ± 0.40 1.38 ± 0.25 1.35 ± 0.21 1.55 ±0.45 1.38 ± 0.18 1.19 ± 0.16 1.78 ± 0.32 1.38* ± 0.16  COUP-TF 0.05 ±0.01 0.22* ± 0.05  0.13 ± 0.03 0.08 ± 0.04 0.06 ± 0.03 0.07 ± 0.02 0.10± 0.06 0.12 0.10 ± 0.05 0.09 ± 0.03 0.07* ± 0.02  0.08 ± 0.02 (biop)n.a. IL-1β 1.02 ± 0.09 0.86 ± 0.10 0.98* ± 0.07  1.31 ± 0.36 1.14 ± 0.281.23 ± 0.23 0.31 ± 0.15 0.06 0.25* ± 0.12  1.13 ± 0.31 1.05 ± 0.27 1.10± 0.21 (epith) n.a. Cx43 1.06 ± 0.15 0.72 ± 0.32 0.95* ± 0.14  1.69 ±0.21 1.06 ± 0.19 1.39* ± 0.16  1.30 ± 0.27 0.26 1.04 ± 0.32 1.61 ± 0.180.98 ± 0.18 1.33 ± 0.14 (epith) n.a. 1.06* ± 0.15  0.72 ± 0.32 0.95 ±0.14 1.69* ± 0.21  1.06 ± 0.19 1.39 ± 0.16 1.30 ± 0.27 0.26 1.04 ± 0.321.61 ± 0.18 0.98 ± 0.18 1.33 ± 0.14 n.a. HSP 70 0.56 ± 0.09 0.30 ± 0.070.42* ± 0.06  1.04 ± 0.28 0.91 ± 0.26 0.98 ± 0.19 2.61 ± 1.01 2.75 ±1.67 2.70 ± 1.10 1.50 ± 0.38 1.78 ± 0.81 1.65* ± 0.46  (epith) HSP 700.26* ± 0.13  1.38 ± 0.44 0.86 ± 0.28 0.80 ± 0.20 0.80 ± 0.31 0.80 ±0.18 1.06 ± 0.33 0.10 0.99 ± 0.31 0.96* ± 0.22  0.71 ± 0.28 0.89 ± 0.18(biop) n.a. HSP 90 0.38 ± 0.08 0.32 ± 0.06 0.36* ± 0.06  0.37 ± 0.060.19 ± 0.03 0.28 ± 0.04 0.05 ± 0.02 n.a. 0.05* ± 0.02  0.30 ± 0.06 0.19± 0.03 0.25 ± 0.04 (epith) n.a. GPx4 (biop) 0.07 ± 0.01 0.21* ± 0.04 0.13 ± 0.02 0.06 ± 0.02 0.07* ± 0.02  0.07 ± 0.01 0.09 ± 0.02 0.81 0.19± 0.10 0.07 ± 0.01 0.15 ± 0.08 0.10 ± 0.03 n.a. GRP78  4.76 ± 0.57 7.97*± 1.75  6.24 ± 0.90 6.49 ± 1.38 1.63 ± 0.71 4.64 ± 1.03 1.66 ± 0.41 4.602.08 ± 0.54 4.97 ± 1.08 1.96* ± 0.71  3.10 ± 0.80 (biop) n.a. cox2(biop) 3.04 ± 0.98 5.88 ± 2.10 4.35* ± 1.12  9.28 ± 1.97 9.73 ± 2.919.46* ± 1.60  2.67 ± 0.79 3.75 2.83 ± 0.68 7.20 ± 1.53 9.07 ± 2.65 7.80± 1.33 n.a.

TABLE 4 Overexpressed endometriosis-related markers ModulatedSignificance Name Specificity Sensitivity phase Modulated EXP group (pvalue)* DD9 (epith) 70% 70% ↑ P

1&2  0.008 DD10 (epith) 71% 71% ↑ P 1 0.04 DD13 (biop) 62% 64% ↑ P&S

2 0.04 DD14 (biop) 78% 58% ↑ P&S 1&2 0.04 DD15 (biop) 85% 79% ↑ P&S 1&2 0.0006 HIF1α (epith) 65% 72% ↑ P&S 1&2  0.028 HIF1α (biop NB) 83% 80% ↑P 1&2  0.019 ARNT (epith) 67% 83% ↑ P 1&2 0.02 Glut-1 (epith) 71% 67% ↑P&S 2  0.037 MnSOD (epith) 76% 58% ↑ P&S 1&2  0.017 MnSOD (biop) 81% 53%↑ P&S 1&2  0.003 GPx (epith) 100%  87.5%   ↑P&S 1&2 1.7 × 10⁻¹¹ ATPsynthase 77% 75% ↑ P&S 1&2 10⁻³  (biop) 93% 64% ↑ P 1&2   8 × 10⁻⁴ c-jun (epith) 75% 67% ↑ S 1 0.05 c-jun (biop) 76% 65% ↑ P&S 1&2  0.005Cx43 (epith) 74% 48% ↑ P&S 1  0.048 77% 64% ↑ P 1 0.02 HSP 70 (epith)73% 61% ↑ P&S 1&2 0.01 HSP 70 (biop) 71% 78% ↑ P 1&2  0.014 cox2 (biop)69% 62% ↑ P&S 1 0.01

TABLE 5 Underexpressed endometriosis-related markers Modulated ModulatedSignificance Name Specificity Sensitivity phase EXP group (p value)* DD1(epith) 75% 90% ↓ S

1 0.01 DD2 (epith) 65% 77% ↓ P

&S 1&2 0.04 DD3 (epith) 63% 69% ↓ P&S 1&2 0.01 DD4 (epith) 83% 85% ↓ S1&2 0.04 DD5 (epith) 78% 79% ↓ P 1&2 0.03 DD5 (biop PROT)+ 77% 61% ↓ S1&2 0.001 71% 57% ↓ S 1&2 0.05 DD6 (epith) 89% 100%  ↓ S 1 0.018 DD7(epith) 80% 70% ↓ P 1&2 0.019 DD8 (epith) 67% 60% ↓ P&S 1 0.02 DD12(biop) 81% 56% ↓ P&S 1&2 0.015 DD12 (biop NB) 55% 68% ↓ P&S 1&2 0.05DD16 (biop) 70% 78% ↓ P&S 1&2 3.5 × 10⁻⁵   AK3 (epith) 68% 82% ↓ P&S 10.005 catalase (biop) 83% 78% ↓ S 1&2 0.001 GST (epith) 54% 74% ↓ P&S 10.026 eNOS (epith) 77% 65% ↓ P 1&2 0.027 CO3/DD11 (epith) 83% 79% ↓ S1&2 0.002 12S rRNA (biop) 77% 67% ↓ P&S 1 0.02 TI227H (biop) 81% 100%  ↓P&S 2 2 × 10⁻⁸ CO2 (biop) 64% 69% ↓ P 1 0.02 aconitase (biop) 80% 86% ↓S 1 0.005 ANT-1 (biop) 86% 74% ↓ P 1&2 0.007 ANT-1 (biop NB) 75% 73% ↓ P1&2 0.016 Bcl-2 (biop) 54% 82% ↓ P&S 1&2 0.01 COUP-TF (biop) 83% 78% ↓ S1&2 0.01 IL-1β (epith) 78% 100%  ↓ P&S 2 0.0002 HSP 90 (epith) 75% 100% ↓ P&S 2 4.3 × 10⁻⁵   GPx4 (biop) 83% 75% ↓ S 1 5 × 10⁻³ GRP78 (biop) 92%78% ↓ S 1&2 6 × 10⁻³

TABLE 6 Endometriosis-related markers modulated in the proliferativephase Modulated Significance Name Specificity Sensitivity phaseModulated EXP group (p value)* DD5 (epith) 78% 79% ↓ P

1&2 0.03  DD7 (epith) 80% 70% ↓ P 1&2 0.019 DD9 (epith) 70% 70% ↑ P 1&20.008 DD10 (epith) 71% 71% ↑ P 1 0.04  HIF1α (biop NB) 83% 80% ↑ P 1&20.019 ARNT (epith) 67% 83% ↑ P 1&2 0.02  eNOS (epith) 77% 65% ↓ P 1&20.027 CO2 (biop) 64% 69% ↓ P 1 0.02  ANT-1 (biop) 86% 74% ↓ P 1&2 0.007ATP synthase 93% 64% ↑ P 1&2   8 × 10⁻⁴ (biop) Cx43 (epith) 77% 64% ↑ P1 0.02  HSP 70 (biop) 71% 78% ↑ P 1&2 0.014 DD2 (epith) 65% 77% ↓ P&S

1&2 0.04  DD3 (epith) 63% 69% ↓ P&S 1&2 0.01  DD8 (epith) 67% 60% ↓ P&S1 0.02  DD12 (biop) 81% 56% ↓ P&S 1&2 0.015 DD12 (biop NB) 55% 68% ↓ P&S1&2 0.05  DD13 (biop) 62% 64% ↑ P&S 2 0.04  DD14 (biop) 78% 58% ↑ P&S1&2 0.04  DD15 (biop) 85% 79% ↑ P&S 1&2  0.0006 DD16 (biop) 70% 78% ↓P&S 1&2 3.5 × 10⁻⁵ HIF1α (epith) 65% 72% ↑ P&S 1&2 0.028 AK3 (epith) 68%82% ↓ P&S 1 0.005 Glut-1 (epith) 71% 67% ↑ P&S 2 0.037 MnSOD (epith) 76%58% ↑ P&S 1&2 0.017 MnSOD (biop) 81% 53% ↑ P&S 1&2 0.003 GPx (epith)100%  87.5%   ↑ P&S 1&2  1.7 × 10⁻¹¹ GST (epith) 54% 74% ↓ P&S 1 0.02612S rRNA (biop) 77% 67% ↓ P&S 1 0.02  TI227H (biop) 81% 100%  ↓ P&S 2  2 × 10⁻⁸ ANT-1 (biop NB) 85% 75% ↓ P&S 1&2 0.016 ATP synthase 77% 75%↑ P&S 1&2 10⁻³   (biop) Bcl-2 (biop) 54% 82% ↓ P&S 1&2 0.01  c-jun(biop) 76% 65% ↑ P&S 1&2 0.005 IL-1β (epith) 78% 100%  ↓ P&S 2  0.0002Cx43 (epith) 74% 48% ↑ P&S 1 0.048 HSP 70 (epith) 73% 61% ↑ P&S 1&20.01  HSP 90 (epith) 75% 100%  ↓ P&S 2 4.3 × 10⁻⁵ cox2 (biop) 69% 62% ↑P&S 1 0.01 

TABLE 7 Endometriosis-related markers modulated in the secretory phaseModulated Modulated Significance Name Specificity Sensitivity phase EXPgroup (p value)* DD1 (epith) 75% 90% ↓ S

1 0.01  DD4 (epith) 83% 85% ↓ S 1&2 0.04  DD6 (epith) 89% 100%  ↓ S 10.018 catalase (biop) 83% 78% ↓ S 1&2 0.001 CO3/DD11 (epith) 83% 79% ↓ S1&2 0.002 aconitase (biop) 80% 86% ↓ S 1 0.005 c-jun (epith) 75% 67% ↑ S1 0.05  COUP-TF (biop) 83% 78% ↓ S 1&2 0.01  GPx4 (biop) 83% 75% ↓ S 1  5 × 10⁻³ GRP78 (biop) 92% 78% ↓ S 1&2   6 × 10⁻³ DD2 (epith) 65% 77% ↓P

&S 1&2 0.04  DD3 (epith) 63% 69% ↓ P&S 1&2 0.01  DD8 (epith) 67% 60% ↓P&S 1 0.02  DD12 (biop) 81% 56% ↓ P&S 1&2 0.015 DD12 (biop NB) 55% 68% ↓P&S 1&2 0.05  DD13 (biop) 62% 64% ↑ P&S 2 0.04  DD14 (biop) 78% 58% ↑P&S 1&2 0.04  DD15 (biop) 85% 79% ↑ P&S 1&2  0.0006 DD16 (biop) 70% 78%↓ P&S 1&2 3.5 × 10⁻⁵ HIF1α (epith) 65% 72% ↑ P&S 1&2 0.028 AK3 (epith)68% 82% ↓ P&S 1 0.005 Glut-1 (epith) 71% 67% ↑ P&S 2 0.037 MnSOD (epith)76% 58% ↑ P&S 1&2 0.017 MnSOD (biop) 81% 53% ↑ P&S 1&2 0.003 GPx (epith)100%  87.5%   ↑ P&S 1&2  1.7 × 10⁻¹¹ GST (epith) 54% 74% ↓ P&S 1 0.02612S rRNA (biop) 77% 67% ↓ P&S 1 0.02  TI227H (biop) 81% 100%  ↓ P&S 2  2 × 10⁻⁸ ANT-1 (biop NB) 85% 75% ↓ P&S 1&2 0.016 ATP synthase 77% 75%↑ P&S 1&2 10⁻³   (biop) Bcl-2 (biop) 54% 82% ↓ P&S 1&2 0.01  c-jun(biop) 76% 65% ↑ P&S 1&2 0.005 IL-1β (epith) 78% 100%  ↓ P&S 2  0.0002Cx43 (epith) 74% 48% ↑ P&S 1 0.048 HSP 70 (epith) 73% 61% ↑ P&S 1&20.01  HSP 90 (epith) 75% 100%  ↓ P&S 2 4.3 × 10⁻⁵ cox2 (biop) 69% 62% ↑P&S 1 0.01 

TABLE 8 Endometriosis-related markers modulated according to the stageof the disease Modulated Modulated Significance Name SpecificitySensitivity phase EXP group (p value)* DD1 (epith) 75% 90% ↓ S

1 0.01 DD6 (epith) 89% 100%  ↓ S 1 0.018 DD8 (epith) 67% 60% ↓ P

&S 1 0.02 DD10 (epith) 71% 71% ↑ P 1 0.04 AK3 (epith) 68% 82% ↓ P&S 10.005 GST (epith) 54% 74% ↓ P&S 1 0.026 12S rRNA (biop) 77% 67% ↓ P&S 10.02 CO2 (biop) 64% 69% ↓ P 1 0.02 aconitase (biop) 80% 86% ↓ S 1 0.005c-jun (epith) 75% 67% ↑ S 1 0.05 Cx43 (epith) 74% 48% ↑ P&S 1 0.048 77%64% ↑ P 1 0.02 GPx4 (biop) 83% 75% ↓ S 1 5 × 10⁻³ cox2 (biop) 69% 62% ↑P&S 1 0.01 DD13 (biop) 62% 64% ↑ P&S 2 0.04 Glut-1 (epith) 71% 67% ↑ P&S2 0.037 TI227H (biop) 81% 100%  ↓ P&S 2 2 × 10⁻⁸ IL-1β (epith) 78% 100% ↓ P&S 2 0.0002 HSP 90 (epith) 75% 100%  ↓ P&S 2 4.3 × 10⁻⁵  

TABLE 9 Endometriosis-related markers overexpressed in stage I or II(EXP 1), or in stage III or IV (EXP 2) the disease Speci- Sensi-Modulated Modulated Significance Name ficity tivity phase EXP group (pvalue)* DD10 (epith) 71% 71% ↑ P 1 0.04 c-jun (epith) 75% 67% ↑ S 1 0.05Cx43 (epith) 74% 48% ↑ P&S 1 0.048 77% 64% ↑ P 1 0.02 cox2 (biop) 69%62% ↑ P&S 1 0.01 DD13 (biop) 62% 64% ↑ P&S 2 0.04 Glut-1 71% 67% ↑ P&S 20.037 (epith)

TABLE 10 Endometriosis-related markers underexpressed in stage I or II(EXP 1), or in stage III or IV (EXP 2) of the disease Speci- Sensi-Modulated Modulated Significance Name ficity tivity phase EXP group (pvalue)* CO2 (biop) 64% 69% ↓ P 1 0.02 DD1 (epith) 75% 90% ↓ S

1 0.01 DD6 (epith) 89% 100%  ↓ S 1 0.018 aconitase 80% 86% ↓ S 1 0.005(biop) GPx4 (biop) 83% 75% ↓ S 1 5 × 10⁻³ DD8 (epith) 67% 60% ↓ P

&S 1 0.02 AK3 (epith) 68% 82% ↓ P&S 1 0.005 GST (epith) 54% 74% ↓ P&S 10.026 125 rRNA 77% 67% ↓ P&S 1 0.02 (biop) TI227H 81% 100%  ↓ P&S 2 2 ×10⁻⁸ (biop) IL-1β (epith) 78% 100%  ↓ P&S 2 0.0002 HSP 90 75% 100%  ↓P&S 2 4.3 × 10⁻⁵   (epith)Legend for Tables 3 to 10:

-   CTL: Control group (endometriosis-free women)-   ENDO: Endo group (women having endometriosis)-   EXP: Refer to the Endo group:    -   EXP1: women at stages I and II of the disease    -   EXP2: women stages III and IV of the disease-   epith: marker derived from epithelial cell RNA-   biop: marker derived from unfractioned biopsy RNA-   biop PROT⁺: marker derived from unfractioned biopsy protein-   *: indicates a significant difference according to the Student t    test of the compared groups described-   S: secretory phase-   P: proliferative phase-   NB: indicates marker derived from unfractioned biopsy RNA obtained    by northern blot analysis-   n.a.: not available

TABLE 11 Combinations of genetic endometriosis-related markersSpecificity Sensitivity Individual markers GRP 78 92% 78% alone Catalase83% 78% COUP-TF 83% 78% Combination of 2 GRP 78 & catalase 75% 100% markers GRP 78 & COUP-TF 75% 89% Catalase & COUP-TF 75% 100% Combination of 3 GRP 78 & catalase & 92% 89% markers COUP-TF

While several embodiments of the invention have been described, it willbe understood that the present invention is capable of furthermodifications, and this application is intended to cover any variations,uses, or adaptations of the invention, following in general theprinciples of the invention and including such departures from thepresent disclosure as to come within knowledge or customary practice inthe art to which the invention pertains, and as may be applied to theessential features hereinbefore set forth and falling within the scopeof the invention or the limits of the appended claims.

What is claimed is:
 1. A method for determining the likelihood ofendometriosis in a female human subject, comprising the steps of:obtaining a sample of endometrial cells from said female human subject;assaying said endometrial cells sample for the expression level of atleast one marker selected from the group consisting of: i) GP_(x) mRNAgene comprising an 819-base polynucleotide sequence encoding glutathioneperoxidase; and ii) glutathione peroxidase; comparing said marker levelin said sample with the marker level found in healthy endometriosis-freesubjects, an elevated level of said marker in said sample over theendometriosis-free level being indicative of the likelihood ofendometriosis in said female human subject.
 2. The method of claim 1,wherein endometrial cells from said female subject are sampled at theproliferative phase of her estrous cycle, and wherein overexpressionlevel of at least one said marker is indicative of a higher likelihoodof endometriosis in said female subject as compared to anendometriosis-free female subject.
 3. The method of claim 1, whereinendometrial cells from said female subject are sampled at the secretoryphase of her estrous cycle, and wherein overexpression level of at leastone said marker is indicative of a higher likelihood of endometriosis insaid female subject as compared to an endometriosis-free female subject.4. The method of claim 1, wherein said endometrial cells are eutopicendometrial cells.
 5. The method of claim 1, wherein said endometrialcells are epithelial endometrial cells.
 6. The method of claim 1,further comprising the step of comparing the level of said at least onesaid endometriosis-related marker to an established baseline level. 7.The method of claim 6, wherein said endometriosis-free level of said atleast one said marker has been established by assaying the level of saidat least one marker in a negative reference group of endometriosis-freewomen.
 8. The method of claim 1 wherein the marker is glutathioneperoxidase.
 9. The method of claim 1 wherein said marker is the GPx mRNAcomprising an 819-base polynucleotide sequence.